WO2016065494A1 - Polypeptide molecules against vascular endothelial growth factor (vegf) - Google Patents

Polypeptide molecules against vascular endothelial growth factor (vegf) Download PDF

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WO2016065494A1
WO2016065494A1 PCT/CL2015/000057 CL2015000057W WO2016065494A1 WO 2016065494 A1 WO2016065494 A1 WO 2016065494A1 CL 2015000057 W CL2015000057 W CL 2015000057W WO 2016065494 A1 WO2016065494 A1 WO 2016065494A1
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antibody
vegf
human
molecules
recombinant
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PCT/CL2015/000057
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Spanish (es)
French (fr)
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Oliberto Sanchez Ramos
Frank Camacho Casanova
Jorge Toledo Alonso
Rodrigo Mansilla Ojeda
Lionel Zapata Vega
Alexis Salas Burgos
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Universidad De Concepcion
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the technology described below is intended for the biotechnology, health and pharmaceutical sector, since it includes the generation of molecules that could be used in the manufacture of drugs for the treatment of pathologies that occur with angiogenesis increase.
  • Angiogenesis is implicated in the pathogenesis of different disorders, including solid tumors, intraocular neovascular syndromes, rheumatoid arthritis and psoriasis 1 .
  • solid tumors the formation of new vessels gives tumor cells an advantage of growth and proliferative autonomy compared to normal cells. Consequently, a correlation between the density of the microvessels in tumor sections and the survival of the patient in breast cancer, as well as in several other tumors, has been observed.
  • VEGF Vascular endothelial growth factor
  • VEGF is a homodimeric glycoprotein formed by two 23 kDa subunits, of which there are 5 monomeric isoforms. These include two isoforms that remain attached to the cell membrane (VEGF 189 and VEGF 206) and three of a soluble nature (VEGF 121, VEGF 145, and VEGF 165).
  • the VEGF 165 isoform is the one that predominates in mammalian tissues, except in lung and heart, where VEGF 189 3 predominates, and in placenta, where VEGF 121 4 expression prevails.
  • VEGF has an important regulatory function in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adulthood. The importance of the role played by VEGF has been demonstrated in studies that show that inhibition of a VEGF allele resulted in the death of the embryo as a result of vasculature development failure 5 .
  • VEGF is able to bind to one of the multiple tyrosine kinase III receptors which causes its autophosphorylation, achieving the activation of kinase proteins with mitogenic action 6 .
  • the VEGF receptors called VEGFRl / flt-1 and VEGF / flk-1.
  • VEGFR2 (KDR / Flkl) receptor mediates the biological effects of VEGF-A, and also binds to VEGF-C and VEGF-D ligands. This receptor is expressed differentially in the active endothelium and in some cell lines of tumor origin where it establishes autocrine loops with the secreted VEGF 7-8.
  • the overexpression of this molecule has been related to the progression of lung cancer 9, 10 , endometrial cancer or malignant mesotheliomas 12 , astrocytic neoplasms 13 , primary breast cancer 14 , gastric cancer intestinal type 15 , of glioblastoma multiforme, anaplastic oligodendrogliomas, and ependymomas with necrosis
  • VEGF vascular endothelial growth factor
  • Therapeutic strategies for cancer are mainly based on blocking angiogenesis by blocking VEGF-A and / or its receptors. These strategies are based on the use of monoclonal antibodies (mAbs), metalloproteinase inhibitors, inducers of apoptosis of the tumor endothelium, ribozymes that decrease the expression of VEGF or its receptors. Of all these molecules, the AcM, or molecules derived from them, are the ones that have had the greatest application and acceptance as therapeutic products. As examples of these therapeutic products we can find Bevacizumab and Ranibizumab. Bevacizumab (Trade name Avastin®) is a chimeric monoclonal antibody that recognizes and neutralizes human VEGF-A 18 . Ranibizumab (trade name: Lucentis®) is an antibody fragment derived from the monoclonal antibody bevacizumab. This antibody fragment has a greater affinity for VEGF and is smaller in size than its parental antibody 19 .
  • the MAs have been widely used in therapeutic treatments in pathologies such as cancer and autoimmune diseases, there are a set of elements that limit their applications, make them more expensive and limit their entry into the commercial phase.
  • the size and complexity of the molecule can be listed of antibody, which increase production costs.
  • the size and complexity of the antibody molecule can be listed, which increase production costs 20 .
  • the size of these molecules interferes with their ability to penetrate solid tumors or cross biological barriers such as the blood brain barrier 21 .
  • Another of the limitations of monoclonal antibodies for therapeutic use is that these antibodies must be human, and since most monoclonal antibodies are of murine origin, it becomes necessary to humanize these molecules by antibody engineering 22 .
  • immunoglobulins prevents these glycoproteins from being produced in simpler microorganisms such as bacteria and yeasts, forcing them to be produced in higher cell culture systems that are expensive, technically demanding, and generally unproductive. All these elements contribute to raising product costs for the final recipient 23 .
  • Phage Display technology as a platform for the generation and subsequent production of antibody fragments is still under development.
  • scFv single-chain antibody fragments
  • dAb human antibody domains
  • the chemical modification is one of the methods widely used to prolong the lifetime of the therapeutic product. It is based primarily on increasing the size of the therapeutic compound by conjugation with natural or synthetic polymers using well established procedures known as PEGylation (chemical conjugation with polyethylene glycol). Another method of modifying these biotherapeutics is through the fusion of long-lived proteins in the plasma such as albumin, immunoglobulins or parts of these proteins 1125 . However, all these modifications often cause a significant reduction in the biological activity of the therapeutic product, which makes it difficult to reach the commercial application stages. With these elements, the search for new means to prolong the half-life in circulation of therapeutic proteins remains a challenging task of current biotechnology.
  • Tetranectin is a C-type trimeric lectin that binds Ca 2+ present in blood plasma and in the extracellular matrix of several tissues.
  • the group of tetranectin proteins comprises molecules of human and murine origin which have homology with lectins type C isolated from cattle and sharks 26 .
  • Mature tetranectin is a polypeptide of 181 amino acids encoded by three exons where exon 3 encodes a subunit with different function and structure. It has been proposed that the trimerization of tetranectin is directed by a peptide encoded in exon l 27 .
  • Human tetranectin is capable of forming very stable trimers that can be used as carrier proteins of molecules of therapeutic interest. This will reduce the clearance in the circulatory system of small-sized therapeutic molecules. References.
  • Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246: 1306-9.
  • VEGF121 a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J Biol Chem 1995; 270: 11322-6.
  • Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma. J Pathol 2001; 193: 468-75.
  • Figure 1 This figure shows the cloning of the hVEGFm gene in the pAdtrack-CMV vector, belonging to the "AdEasyTM Adenoviral Vector System” kit, from Agilent Technologies.
  • Figure 2 Results of the purification of the recombinant hVEGF121 protein from the supernatant of Sil-la cells infected with the adenovirus carrying the gene of this protein.
  • Figure 3 Proliferation of HUVEC cells treated with increasing amounts of hVEGF121 produced in our laboratory.
  • Figure 4 Pro-angiogenic activity n / n lived from the purified hVEGF121.
  • FIG. 5 Estimation of the affinity of the phage clones bearing single stranded antibody fragments (scFv) selected against hVEGF121.
  • Figure 6 Scheme of the plasmid pPSIO used for the production of trimeric molecules formed by scFvH2V-DTetr, in the culture supernatant of Pichia pastoris.
  • Figure 7 Structural model of the scFvH2V-DTetr trimeric molecule.
  • A Side view
  • B Top view.
  • Figure 8 Purification of the trimeric molecule H2V-DTetr from Pichia pastoris cell culture medium.
  • Figure 9 Capacity of the trimeric molecule H2V-DTetr to interfere with the stimulatory effect of the hVEGFm molecule.
  • the present invention describes recombinant polypeptide molecules related to antibodies, which specifically recognize the Human Vascular Endothelium Growth Factor (VEGF-A), and interfere with its stimulatory effects in vitro, and pro-angiogenic in vivo.
  • VEGF-A Human Vascular Endothelium Growth Factor
  • These molecules comprise the variable regions of human immunoglobulins that recognize human VEGF-A epitopes and block the pro-angiogenic effect of this molecule.
  • These polypeptide molecules are formed by several sites, composed of amino acids encoded by DNA sequences of variable regions of antibodies, binding to VEFG-A.
  • the molecules can be recombinant antibodies, single chain antibody fragments (scFv), Fab type antibody fragments, or multimeric molecules. Such recombinant polypeptide molecules are capable of inhibiting VEGF-induced proliferation of human endothelial cells in in vitro assays.
  • These molecules can form trimers when fused to the trimerization domain of human tetranectin and can be used for passive immunotherapy of pathological entities whose course is associated with increased vasculature, such as age-related macular degeneration (wet variant), the cancer and its metastases, neovascular glaucoma, diabetic and newborn retinopathy, acute and chronic inflammatory processes, infectious diseases, autoimmune diseases, organ transplant rejection, hemangiomas and angiofibromas, among others.
  • pathological entities whose course is associated with increased vasculature, such as age-related macular degeneration (wet variant), the cancer and its metastases, neovascular glaucoma, diabetic and newborn retinopathy, acute and chronic inflammatory processes, infectious diseases, autoimmune diseases, organ transplant rejection, hemangiomas and angiofibromas, among others.
  • polypeptide molecules are fused to the trimerization domain of human tetranectin or any of the sequences belonging to the family of type C lectins, which allows it to increase its half-life in circulation.
  • the present invention describes a new approach to increase the half-life in circulation of recombinant proteins of therapeutic value.
  • the invention also describes multimeric molecules composed of at least two monomeric polypeptide constructs and further describes the methods of preparing these gene constructs and polypeptides. These multimeric molecules are for therapeutic use, such as tumor treatment by inhibiting the pro-angiogenic properties of VEGF or any other therapy using polypeptides related to antibody molecules.
  • This invention describes any combination of antibody-related polypeptide molecules, not necessarily with recognition by the same epitope, with the trimerization domain of human tetranectin.
  • polypeptides derived from immunoglobulins partially or totally synthetically produced (via recombinant deoxyribonucleic acid (DNA) or artificial gene synthesis) with specific recognition of an antigen through one or more domains that interact with it, formed by particular combinations of variable regions of heavy and light chains of immunoglobulins, and which is commonly referred to as the antigen binding site.
  • recombinant antibodies are the so-called single chain antibody (scFv) fragments obtained by genetic engineering comprising one or more antigen binding sites.
  • VH and VL domains of a single antibody bind in different sequence (VH-VL or VL-VH) with a peptide binding segment (linker) that allows the two domains to associate to form an antigen binding site .
  • linker a peptide binding segment
  • dAb antibody domains
  • Antibody fragments can be obtained from antibody libraries, where a broad repertoire of genes (whether synthetic, semi-synthetic or natural) from the variable regions of a species are randomly combined to produce particular associations of variable regions of antibodies, which are then exposed on the surface of filamentous phages.
  • the first term describes the part of an antibody that interacts specifically with an antigen (or part of it).
  • an antibody can bind only to a particular part of the antigen, whose part is called an epitope.
  • An antibody binding site is primarily formed by two antibody variable regions, the light chain variable region and the heavy chain variable region.
  • the antibody binding site is formed by the non-covalent interaction of the variable regions.
  • the binding site of an antibody can be artificially stabilized by binding the two variable regions with a binding peptide (linker) that does not interfere with its antigen-specific recognition properties. This is the case of a scFv type fragment.
  • the epitope recognized by the binding site of an antibody in the case of being the antigen a protein, can be formed by a linear sequence of amino acids, or be conformational, it being understood that the amino acids recognized by the binding site of the antibody they are close in the tertiary structure of the protein, but not They are necessarily sequential in their primary structure.
  • the epitope is by nature a discrete zone, defined by a particular group of amino acids that interact with those of the antibody through non-covalent bonds.
  • polypeptide region of the human tetranectin molecule or any member of the family of type C lectins described, whatever their origin.
  • region between amino acids 47 and 73 of the human tetranectin A chain (NCBI: CAA45860) is used as the trimerization domain.
  • the sequence comprises a fragment with an alpha helix structure and the following amino acid sequence: LKS LDTLSQEVALLKEQQALQTVCLK) (SEQID7)
  • the antibody fragments described in this invention specifically recognize isotherms 121, 148, 165 and 189 of human VEGF.
  • the scFv-like antibody fragments are: H2V (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), and DI 1 VII (SEQID6), these were selected from one semi-synthetic scFv library deployed in filamentous phages.
  • Another type of molecule is the antibody fragment that is composed of the sequences H2V, (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), and D11VII (SEQID6) fused to trimerization domain of human tetranectin. (SEQID7) and separated by a spacer sequence (SEQID8).
  • VEGF-A or any antigen
  • they may be in the form of bi-specific antibody molecules, where a portion thereof retains its specificity for human VEGF-A (or any antigen) and another has a different specificity. All these manipulations by genetic engineering are known to those skilled in the art.
  • SEQID7 Tetranectin domain aa 47-73 chain A.
  • Example 1 Cloning and expression in SiHa cells and purification of isoform 121 of VEGF.
  • the human VEGF 121 sequence was obtained from the NCBI databases (GenBank: AAF19659.1). Being the smallest, all epitopes present in this variant, are also contained in the other isoforms of human VEGF. This ensures that the antibodies, generated against variant 121, will also be able to recognize variants of VEGF 189 and 206. In order to facilitate their subsequent detection and purification, it was decided to incorporate a tail of 6 histidine residues (6HIS ) at one end of the hVEGFm molecule.
  • 6HIS 6 histidine residues
  • the gene coding for the hVEGFm variant was synthesized by the North American company Blu Heron. To facilitate subsequent cloning steps, the hVEGFm gene was flanked by restriction sites Xho I and EcoR V. By digestion with these enzymes, the band corresponding to the hVEGF 121 gene was extracted and cloned into the adenoviral transfer vector pAdTrack -CMV, belonging to the "AdEasyTM kit Adenoviral Vector System ", by Agilent Technologies.
  • FIG. 2A shows the process chromatogram.
  • Figure 2B shows electrophoresis in 12.5% pliacrylamide gels where they are observed from left to right: entry medium, unbound proteins, washed with 80 mM imidaziol, and elution with 250 mM imidazole. For chromatography, imidazole was added to the culture medium until it reached a concentration of 5 mM.
  • the matrix was washed with 80 mM imidazole in phosphate buffer pH 7.2, and a simple elution step was performed in 250 mM imidazole.
  • the fractions were analyzed by electrophoresis, under reducing conditions, of each of the chromatography stages.
  • the purification process yielded two bands within the range of 15 to 20 kDa that appear to be the glycosylated and non-glycosylase forms of hVEGFm.
  • n / n sitW activity a primary culture of HUVEC cells was performed (step 4).
  • the cells were cultured in 96-well plates at a rate of 10,000 cells per well.
  • the activity of hVEGF121 in the concentration range between 0.1 and 1000 ng / ml was evaluated.
  • the cells were stained with MTT (3- (4, 5- dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide), and the absorbance was read at 570 nm.
  • MTT 4- (4, 5- dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide
  • the proangiogenic activity of hVEGF 121 was determined by an n / n vivd 'angiogenesis assay using matrigel (BD).
  • An experimental group was made where the matrigel mixture contained heparin (64 IU / mL) and hVEGFm (30 ng / mL). Of this mixture, 300 ⁇ _ were injected subcutaneously into three CFL-BALB / c hybrid mice. As a negative control, three mice were used to inject a mixture of matrigel and heparin, lacking hVEGFm. Seven days after the injection, the mice were sacrificed and a matrigel plug was removed.
  • Figure 4 shows these results mice inoculated with matrigel, with (A) or without purified hVEGF 121 (B) (30 ng / mL).
  • Figure 4C shows the quantification of neovascularization in matrigel plugs by measuring hemoglobin content.
  • the arrow shows the formation of blood vessels inside the matrigel plug.
  • B Matrix inoculated mouse lacking hVEGF121. No glasses are observed inside the cap.
  • C the neovascularization in the matrigel caps was quantified by measuring the hemoglobin content in the cap with the Drabkin method, the results are shown as the mean ⁇ SD of three caps. The results obtained suggest that the hVEGF produced by this methodology is biologically active.
  • Example 2 Selection of high affinity antibody fragments against human VEGF.
  • scFv single chain antibody fragments
  • CDR complementarity determining regions
  • the human hVEGFi 2 i protein carrying the 6HIS tag was used as an antigen.
  • Nickel-coated plates were used to capture the 6HIS tag of which this human VEGF variant is a carrier.
  • the eluted phages were amplified in the TG1 strain of the cofi were used as starting material in the next selection cycle. This procedure was repeated 3 times under the same conditions. Subsequently, individual colonies of TG1 infected with the eluted phages of the second and third selection cycle were randomly selected to produce phages at 96-well scale. The ability of these phage clones that carry scFv antibody fragments to bind VEGF was evaluated by ELISA. For this, Maxisorp 96-well plates were coated with 5 pg / mL of VEGF, and then blocked.
  • the phages diluted in PBS-4% skim milk were incubated for 1 hour at 25 ° C in the plates, followed by several washes. Bound phages were detected by the addition of peroxidase-conjugated anti-M13 antibodies (Amersham) for 1 hour at 25 ° C. After several washes, the reaction was developed with the addition of peroxidase substrate solution (TMB). The absorbance was read at 450 nm in a microplate reader. Of the 90 clones evaluated, 57 were positive.
  • Example 3 Selection of clones by estimating their affinities for VEGF.
  • the affinities were estimated using a single-point competitive ELISA.
  • the phages diluted in PBS-Tween with or without 100 nM VEGF were incubated for 1 hour at 25 ° C.
  • the mixtures were transferred to Maxisorp ELISA plates (Nunc) coated with VEGF and incubated for 15 minutes at the same temperature, followed by several washes.
  • Bound phages were detected by the addition of peroxidase-conjugated anti-M13 antibodies (Amersham) for 1 hour at 25 ° C. After several washes, the reaction was developed with the addition of peroxidase substrate solution (TMB).
  • TMB peroxidase substrate solution
  • Example 4 Cloning of scFv H2V-DTetr fragment in the pPSIO vector.
  • the pPSIO vector is a plasmid designed for expression in P. Pastoris yeast.
  • This vector contains as main elements the origin of replication and the gene of ⁇ -lactamase of E col /, a regulatory fragment of the promoter of the AOX1 gene of P. pastoris, the secretion signal of the sucrose invertase enzyme (ssSUC2) , encoded by the S. cerevisiae SUC2 gene, the transcription terminator of the S. cerevisiae glyceraldehyde 3 phosphate dehydrogenase (tGAP) gene, the 5. cerevisiae HIS3 gene and the 3 'region of the AOX1 gene of P. pastoris ( Figure 6).
  • This molecule contains the amino acid sequence of the H2V clone of the scFv-like antibody fragment that recognizes the human VEGF molecule isoform 121. This fragment is linked through a peptide sequence to the trimerization domain of human tetranectin. (SEQID7). This sequence, which ranges from amino acid 42 to 73 of the human tetranectin of the A chain, is what makes it possible to form the scFvH2V-DTetr trimers.
  • Table 1 Sequence of the scFvH2V-DTetr molecule.
  • the coding sequence corresponding to the H2V clone fused to the trimerization domain of human tetranectin (DTetr) was obtained by chemical synthesis to which the recognition sites for Smal and Nael restriction endonucleases were added. This gene was provided by the GenScript company in the commercial plasmid pUC-K ( Figure 6).
  • the trimerization domain of tetranectin formed by the amino acid sequence (43-73) of human tetranectin allows the formation of the trimeric molecule formed by scFv H2V-DTetr fusion protein monomers ( Figure 7)
  • the pPSIO vector was digested with the restriction enzymes Smal and Nael. The ends of the linear vector were subsequently dephosphorylated with Antarctic phosphatase.
  • the plasmid containing the scFv H2V-DTetr fragment was digested with the enzymes Smal and Nael and purified next to the vector from a 0.8% low melting agarose gel.
  • the fragments of interest were ligated with the enzyme T4 DNA ligase, the reaction was transformed into E coi / cells and the recombinant plasmids were detected by restriction assays with the same enzymes.
  • the resulting expression plasmid was called pPscFvH2V-Dtetr.
  • Example 5 Transformation, expression and purification of the multimeric protein pPscFvH2V-DTetr in Pichia pastoris.
  • Induction experiments were performed in Erlenmeyer flasks of 1 L capacity. It was split with a pre-inoculum of 10 ml_ prepared in YPG culture medium, this was grown for 20 hours in orbital shaking (240 rpm) at 28 ° C. From the pre-inoculum already grown, a 500 ml culture of YP medium supplemented with 2% glycerol was inoculated. After 20 hrs the medium was centrifuged at 3,500 rpm for 5 min, the supernatant was removed and the pellet was resuspended in 500mL of YP medium.
  • Induction of scFvH2V-DTetr protein expression was started immediately by supplementing the YP medium with 1% Methanol every 12 hours keeping the batch culture at 28 ° C in orbital shaking (240 rpm).
  • the optical density (600 nm) of the culture was measured every 24 hours to study the growth kinetics during induction.
  • the 500mL of culture medium was centrifuged at 6200 rpm for 5 minutes, the supernatant was recovered which was centrifuged at 8,000 rpm for 15 minutes, the supernatant was clarified through a fiberglass pre-filter and finally filtered at 0.45 -m.
  • the clarified medium was subjected to ultrafiltration in nitrocellulose membrane with an exclusion size of lOKDa.
  • the medium was concentrated and during this process it was replaced with 150 mL of 50mM phosphate buffer pH 7.0.
  • a total of 15 ml of clarified and concentrated culture supernatant was purified by metal ion affinity chromatography, for which the TOYOPEARL chelate resin (TOYO, Japan) was used.
  • the inlet sample was loaded at pH 8.0 with 5 mM Imidazole, allowed to flow by gravity at room temperature, nonspecific protein binding was removed by a 50 mM imidazole wash step in phosphate buffer at pH 7, 5, Final elution of the protein was performed at 250 mM Imidazole in phosphate buffer pH7.0.
  • the input fractions, unbound sample, washing and elution obtained were collected of the purification process. These fractions were subjected to protein electrophoresis in acrylamide and Westen-Blot gels under non-denaturing conditions.
  • Example 6 Test of the inhibition of HUVEC cell proliferation by the trimer formed by scFv H2V-DTetr.
  • HUVEC cells (4th pass) were seeded in 96-well plates (10 3 cells / well) in RPMI-1640 medium, 2% Bovine fetal serum, heparin (50 ug / mL). The cells were incubated for 16 hours at 37 ° C, 5% C02. After incubation, 2 washes were performed with RPMI_1640 medium without serum, and serum-free RPMI medium containing purified hVEGF 121 (10 ng / mL) and increasing amounts of the purified H2V-DTetr trimeric antibody was added to the cells. After a 72 hour incubation at 37 ° C the culture medium was removed and the cells stained with a solution of 0.5% crystal violet and 20% methanol. The inhibition effect was obtained by reading the plates at 562 nm. Cell growth was taken as 100% proliferation under conditions with hVEGF 121 without the trimeric antibody (Figure 9).

Abstract

The invention relates to recombinant antibody-related polypeptide molecules which specifically recognise human vascular endothelial growth factor-A (VEGF-A) and interfere with the in vivo proangiogenic and in vitro stimulatory effects thereof. These polypeptide molecules are formed by multiple sites made up of amino acids encoded by DNA sequences of variable regions of antibodies, for binding to VEFG-A. The molecules can be recombinant antibodies, single-chain antibody fragments (scFv), Fab-type antibody fragments, or multimeric molecules.

Description

MOLÉCULAS POLIPEPTÍDICAS CONTRA EL FACTOR DE CRECIMIENTO DEL ENDOTELIO VASCULAR (VEGF).  POLYPEPTIDE MOLECULES AGAINST THE GROWTH FACTOR OF THE VASCULAR ENDOTELIUM (VEGF).
SECTOR TECNICO TECHNICAL SECTOR
La tecnología que a continuación se describe está destinada al sector biotecnológico, salud y farmacéutico, ya que comprende la generación de moléculas que se podrían utilizar en la fabricación de medicamentos para el tratamiento de patologías que cursan con incremento de la angiogénesis.  The technology described below is intended for the biotechnology, health and pharmaceutical sector, since it includes the generation of molecules that could be used in the manufacture of drugs for the treatment of pathologies that occur with angiogenesis increase.
TECNICA ANTERIOR PREVIOUS TECHNIQUE
La Angiogénesis está implicada en la patogénesis de diferentes trastornos entre los cuales se incluyen los tumores sólido, síndromes neovasculares intraoculares, artritis reumatoide y soriasis1. En el caso de los tumores sólidos, la formación de nuevos vasos le confiere a las células tumorales una ventaja de crecimiento y autonomía proliferativa en comparación con las células normales. En consecuencia, se ha observado una correlación entre la densidad de los microvasos en secciones de tumor y la supervivencia del paciente en el cáncer de mama, así como en varios otros tumores. Angiogenesis is implicated in the pathogenesis of different disorders, including solid tumors, intraocular neovascular syndromes, rheumatoid arthritis and psoriasis 1 . In the case of solid tumors, the formation of new vessels gives tumor cells an advantage of growth and proliferative autonomy compared to normal cells. Consequently, a correlation between the density of the microvessels in tumor sections and the survival of the patient in breast cancer, as well as in several other tumors, has been observed.
El factor de crecimiento del endotelio vascular (VEGF) pertenece a una familia de moléculas que inducen la formación de nuevos vasos de manera directa y específica 2. Vascular endothelial growth factor (VEGF) belongs to a family of molecules that induce the formation of new vessels directly and specifically 2 .
El VEGF es una glicoproteína homodimérica formada por dos subunidades de 23 kDa, de la cual existen 5 isoformas monoméricas. Estas incluyen dos isoformas que se mantienen unidas a la membrana celular (VEGF 189 y VEGF 206) y tres de naturaleza soluble (VEGF 121, VEGF 145, y VEGF 165). La isoforma VEGF 165 es la que predomina en tejidos de mamíferos, excepto en pulmón y corazón, donde predomina el VEGF 189 3, y en placenta, donde prevalece la expresión del VEGF 1214. VEGF is a homodimeric glycoprotein formed by two 23 kDa subunits, of which there are 5 monomeric isoforms. These include two isoforms that remain attached to the cell membrane (VEGF 189 and VEGF 206) and three of a soluble nature (VEGF 121, VEGF 145, and VEGF 165). The VEGF 165 isoform is the one that predominates in mammalian tissues, except in lung and heart, where VEGF 189 3 predominates, and in placenta, where VEGF 121 4 expression prevails.
El VEGF tiene una importante función regulatoria en la formación de nuevos vasos sanguíneos durante la vasculogénesis embrionaria y en la angiogénesis durante la vida adulta. La importancia del papel desempeñado por el VEGF ha sido demostrada en estudios que muestran que la inhibición de un alelo del VEGF resultó en la muerte del embrión como consecuencia del fallo del desarrollo de la vasculatura5. El VEGF es capaz de unirse a uno de los múltiples receptores de tirosina kinasa III lo que provoca su autofosforilación, consiguiendo la activación de proteínas kinasas con acción mitogénica6. En las células endoteliales se encuentran los receptores de VEGF, denominados VEGFRl/flt-1 y VEGF/flk-1. El receptor VEGFR2 (KDR/Flkl) media los efectos biológicos del VEGF-A, y también se une a los ligandos VEGF-C y VEGF-D. Este receptor se expresa de manera diferencial en el endotelio activo y en algunas líneas celulares de origen tumoral donde establece lazos autocrinos con el VEGF secretado7- 8. VEGF has an important regulatory function in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adulthood. The importance of the role played by VEGF has been demonstrated in studies that show that inhibition of a VEGF allele resulted in the death of the embryo as a result of vasculature development failure 5 . VEGF is able to bind to one of the multiple tyrosine kinase III receptors which causes its autophosphorylation, achieving the activation of kinase proteins with mitogenic action 6 . In the endothelial cells are the VEGF receptors, called VEGFRl / flt-1 and VEGF / flk-1. The VEGFR2 (KDR / Flkl) receptor mediates the biological effects of VEGF-A, and also binds to VEGF-C and VEGF-D ligands. This receptor is expressed differentially in the active endothelium and in some cell lines of tumor origin where it establishes autocrine loops with the secreted VEGF 7-8.
La sobre-expresión de esta molécula se ha relacionado con el avance, del cáncer de pulmón9, 10, cáncer de endometrio u, de mesoteliomas malignos 12, de neoplasmas astrocíticos13, de cáncer primario de mama 14, de cáncer gástrico tipo intestinal 15, de glioblastoma multiforme, oligodendrogliomas anaplásticos, y ependimomas con necrosisThe overexpression of this molecule has been related to the progression of lung cancer 9, 10 , endometrial cancer or malignant mesotheliomas 12 , astrocytic neoplasms 13 , primary breast cancer 14 , gastric cancer intestinal type 15 , of glioblastoma multiforme, anaplastic oligodendrogliomas, and ependymomas with necrosis
16 16
La sobre-expresión del VEGF se ha asociado, además, a la enfermedad autonómica VHL y a hemangioblastomas, al avance de la retinopatía diabética 17 y, junto al Flt-1, a las reacciones de hipersensibilidad retardada. Overexpression of VEGF has also been associated with autonomic VHL disease and hemangioblastomas, with the progression of diabetic retinopathy 17 and, together with Flt-1, with delayed hypersensitivity reactions.
Las estrategias terapéuticas para el cáncer se basan principalmente en el bloqueo de la angiogénesis mediante el bloqueo del VEGF-A y/o sus receptores. Estas estrategias están basadas en el uso de Anticuerpos monoclonales (AcM), inhibidores de metaloproteinasas, inductores de apoptosis del endotelio tumoral, ribozimas que disminuyen la expresión de VEGF o de sus receptores. De todas estas moléculas, los AcM, o moléculas derivadas de estos, son los que mayor aplicación y aceptación han tenido como productos terapéuticos. Como ejemplos de estos productos terapéuticos podemos encontrar el Bevacizumab y el Ranibizumab. El bevacizumab (Nombre comercial Avastin®) es un anticuerpo monoclonal quimérico que reconoce y neutraliza el VEGF-A humano18. El Ranibizumab (nombre comercial: Lucentis®) es un fragmento de anticuerpo derivado del anticuerpo monoclonal bevacizumab. Este fragmento de anticuerpo tienen mayor afinidad por el VEGF y es de menor talla que su anticuerpo parental19. Therapeutic strategies for cancer are mainly based on blocking angiogenesis by blocking VEGF-A and / or its receptors. These strategies are based on the use of monoclonal antibodies (mAbs), metalloproteinase inhibitors, inducers of apoptosis of the tumor endothelium, ribozymes that decrease the expression of VEGF or its receptors. Of all these molecules, the AcM, or molecules derived from them, are the ones that have had the greatest application and acceptance as therapeutic products. As examples of these therapeutic products we can find Bevacizumab and Ranibizumab. Bevacizumab (Trade name Avastin®) is a chimeric monoclonal antibody that recognizes and neutralizes human VEGF-A 18 . Ranibizumab (trade name: Lucentis®) is an antibody fragment derived from the monoclonal antibody bevacizumab. This antibody fragment has a greater affinity for VEGF and is smaller in size than its parental antibody 19 .
Aunque los AcM han sido ampliamente utilizados en los tratamientos terapéuticos en patologías como el cáncer y enfermedades autoinmunes, existen un conjunto de elementos que limitan sus aplicaciones, encarecen su uso y limitan su entrada a la fase comercial. Entre las que se pueden enumerar el tamaño y la complejidad de la molécula de anticuerpo, los cuales aumentan los costos de producción. Entre las que se pueden enumerar el tamaño y la complejidad de la molécula de anticuerpo, los cuales aumentan los costos de producción20. Además el tamaño de estas moléculas interfiere en su capacidad para penetrar tumores sólidos o atravesar barreras biológicas como la barrera hematoencefálica21. Otra de las limitaciones de los anticuerpos monoclonales para el uso terapéutico es que estos anticuerpos deben ser humanos, y como la mayoría de los anticuerpos monoclonales son de origen murino, se hace necesario humanizar estas moléculas mediante ingeniería de anticuerpos22. La complejidad estructural de las inmunoglobulinas impide que estas glicoproteínas puedan ser producidas en microorganismos más sencillos como bacterias y levaduras, obligando a producirlos en sistemas de cultivo de células superiores que resultan costosos, técnicamente exigentes, y generalmente poco productivos. Todas estos elementos contribuyen a elevar los costos del producto para el destinatario final23. Although the MAs have been widely used in therapeutic treatments in pathologies such as cancer and autoimmune diseases, there are a set of elements that limit their applications, make them more expensive and limit their entry into the commercial phase. Among which the size and complexity of the molecule can be listed of antibody, which increase production costs. Among which the size and complexity of the antibody molecule can be listed, which increase production costs 20 . In addition, the size of these molecules interferes with their ability to penetrate solid tumors or cross biological barriers such as the blood brain barrier 21 . Another of the limitations of monoclonal antibodies for therapeutic use is that these antibodies must be human, and since most monoclonal antibodies are of murine origin, it becomes necessary to humanize these molecules by antibody engineering 22 . The structural complexity of immunoglobulins prevents these glycoproteins from being produced in simpler microorganisms such as bacteria and yeasts, forcing them to be produced in higher cell culture systems that are expensive, technically demanding, and generally unproductive. All these elements contribute to raising product costs for the final recipient 23 .
Frente a estas limitaciones de la tecnología convencional, han surgido las bibliotecas combinatorias basadas en las plataformas de Phage Display, que facilitan la obtención de moléculas de afinidad totalmente humanas con capacidad de reconocimiento y unión similares a la de los AcM. La tecnología de Phage Display como plataforma para la generación y posterior producción de fragmentos de anticuerpos aun se encuentra en desarrollo. Existen elementos dentro de esta plataforma que son susceptibles de optimización y pueden mejorar ostensiblemente el rendimiento de la plataforma; entre ellos: (I) la posibilidad de generar moléculas de afinidad con un tiempo de vida media prologando sin tener que renunciar a la producción en microorganismos (bacterias y levaduras); (II) la posibilidad de disminuir la librería de síntesis inicial de 100 millones de combinaciones posibles a 10,000 y el incremento racional de la afinidad de los fragmentos de anticuerpos seleccionados utilizando diseño ¡n silico de la combinatoria inicial e incremento de la afinidad por mutaciones racionales asistidas por modelación estructural y docking molecular de los blancos.24. Faced with these limitations of conventional technology, combinatorial libraries based on Phage Display platforms have emerged, which facilitate the acquisition of fully human affinity molecules with recognition and binding capacity similar to that of the AcM. Phage Display technology as a platform for the generation and subsequent production of antibody fragments is still under development. There are elements within this platform that are susceptible to optimization and can significantly improve the performance of the platform; among them: (I) the possibility of generating affinity molecules with a half-life extending without having to give up the production in microorganisms (bacteria and yeasts); (II) the possibility of reducing the initial synthesis library from 100 million possible combinations to 10,000 and the rational increase in the affinity of the selected antibody fragments using a unique silico design of the initial combinatorial and increased affinity for rational mutations assisted by structural modeling and molecular docking of the targets. 24 .
Mediante la tecnología de Phage Display se pueden diseñar y construir bibliotecas humanas de fragmentos de anticuerpos de simple cadena (scFv) y de dominios de anticuerpos humanos (dAb) de las cuales se pueden obtener moléculas de alta afinidad contra la diana deseada.22. Using Phage Display technology, human libraries of single-chain antibody fragments (scFv) and human antibody domains (dAb) can be designed and constructed from which high affinity molecules against the desired target can be obtained. 22
Las moléculas de menor tamaño derivadas de anticuerpos completos tales scFv o los dAb son rápidamente eliminados del sistema circulatorio, mientras que las moléculas de inmunoglobulinas permanecen por un periodo mayor. Por el contrario, estos fragmentos de anticuerpos exhiben mejores propiedades de extravasación. Por lo que estas moléculas pueden ser optimizadas usando vehículos para su oligomerización controlada. Smaller molecules derived from whole antibodies such scFv or dAb are rapidly removed from the circulatory system, while the Immunoglobulin molecules remain for a longer period. On the contrary, these antibody fragments exhibit better extravasation properties. So these molecules can be optimized using vehicles for controlled oligomerization.
Durante la última década se han desarrollado una amplia gama de productos terapéuticos, a pesar de esto, uno de los objetivos principales del desarrollo de terapias es aumentar el tiempo de vida media en circulación de estos productos. La modificación química es uno de los métodos ampliamente utilizado para prolongar el tiempo de vida del producto terapéutico. Se basa fundamentalmente en el aumento del tamaño del compuesto terapéutico por conjugación con polímeros naturales o sintéticos utilizando procedimientos bien establecidos conocidos como PEGilación (conjugación química con polietilenglicol). Otro método de modificación de estos bioterápicos es a través de la fusión a proteínas de larga vida en circulación en el plasma como la Albúmina, Inmunoglobulinas o partes de estas proteínas1125. Sin embargo, todas estas modificaciones a menudo causan una reducción significativa de la actividad biológica del producto terapéutico lo que dificulta su arribo a las etapas de aplicación comercial. Con estos elementos, la búsqueda de nuevos medios para prolongar el tiempo de vida media en circulación de proteínas terapéuticas, sigue siendo una tarea desafiante de la biotecnología actual. During the last decade a wide range of therapeutic products have been developed, despite this, one of the main objectives of the development of therapies is to increase the average life time in circulation of these products. The chemical modification is one of the methods widely used to prolong the lifetime of the therapeutic product. It is based primarily on increasing the size of the therapeutic compound by conjugation with natural or synthetic polymers using well established procedures known as PEGylation (chemical conjugation with polyethylene glycol). Another method of modifying these biotherapeutics is through the fusion of long-lived proteins in the plasma such as albumin, immunoglobulins or parts of these proteins 1125 . However, all these modifications often cause a significant reduction in the biological activity of the therapeutic product, which makes it difficult to reach the commercial application stages. With these elements, the search for new means to prolong the half-life in circulation of therapeutic proteins remains a challenging task of current biotechnology.
La tetranectina es una lectina trimérica tipo C que une Ca2+ presente en el plasma sanguíneo y en la matriz extracelular de varios tejidos. El grupo de proteínas tetranectinas comprende moléculas de origen humano y murino las cuales presentan homología con lectinas tipo C aisladas a partir de ganado bovino y tiburones26. La tetranectina madura es un polipéptido de 181 aminoácidos codificados por tres exones donde el exón 3 codifica para una subunidad con función y estructura diferente Se ha propuesto que la trimerización de la tetranectina está dirigida por un péptido codificado en el exón l27. Tetranectin is a C-type trimeric lectin that binds Ca 2+ present in blood plasma and in the extracellular matrix of several tissues. The group of tetranectin proteins comprises molecules of human and murine origin which have homology with lectins type C isolated from cattle and sharks 26 . Mature tetranectin is a polypeptide of 181 amino acids encoded by three exons where exon 3 encodes a subunit with different function and structure. It has been proposed that the trimerization of tetranectin is directed by a peptide encoded in exon l 27 .
La tetranectina humana es capaz de formar trímeros muy estables que puede ser usados como proteínas transportadoras (carriers) de moléculas de interés terapéutico. De esta forma se reducirá el aclaramiento en el sistema circulatorio de moléculas terapéuticas de pequeña talla. Referencias. Human tetranectin is capable of forming very stable trimers that can be used as carrier proteins of molecules of therapeutic interest. This will reduce the clearance in the circulatory system of small-sized therapeutic molecules. References.
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Figure imgf000006_0001
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Figure imgf000006_0001
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BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1: Esta figura muestra el clonamiento del gen hVEGFm en el vector pAdtrack- CMV, perteneciente al kit "AdEasyTM Adenoviral Vector System", de Agilent Technologies. Figura 2: Resultados de la purificación de la proteína hVEGF121 recombinante a partir del sobrenadante de células Sil-la infectadas con el Adenovirus portador del gen de esta proteína. Figure 1: This figure shows the cloning of the hVEGFm gene in the pAdtrack-CMV vector, belonging to the "AdEasyTM Adenoviral Vector System" kit, from Agilent Technologies. Figure 2: Results of the purification of the recombinant hVEGF121 protein from the supernatant of Sil-la cells infected with the adenovirus carrying the gene of this protein.
Figura 3: Proliferación de células HUVEC tratadas con cantidades crecientes de hVEGF121 producido en nuestro laboratorio.  Figure 3: Proliferation of HUVEC cells treated with increasing amounts of hVEGF121 produced in our laboratory.
Figura 4: Actividad pro-angiogénica n/n vivó' del hVEGF121 purificado. Figure 4: Pro-angiogenic activity n / n lived from the purified hVEGF121.
Figura 5: Estimación de la afinidad de los clones de fagos portadores de fragmentos de anticuerpo de simple cadena (scFv) seleccionados contra el hVEGF121.  Figure 5: Estimation of the affinity of the phage clones bearing single stranded antibody fragments (scFv) selected against hVEGF121.
Figura 6: Esquema del plasmidio pPSIO empleado para la producción de moléculas triméricas formadas por scFvH2V-DTetr, en el sobrenadante de cultivos de Pichia pastoris.  Figure 6: Scheme of the plasmid pPSIO used for the production of trimeric molecules formed by scFvH2V-DTetr, in the culture supernatant of Pichia pastoris.
Figura 7: Modelo estructural de la molécula trimérica scFvH2V-DTetr. (A) Vista lateral, (B) Vista superior.  Figure 7: Structural model of the scFvH2V-DTetr trimeric molecule. (A) Side view, (B) Top view.
Figura 8: Purificación de la molécula trimérica H2V-DTetr a partir medio de cultivo de células de Pichia pastoris.  Figure 8: Purification of the trimeric molecule H2V-DTetr from Pichia pastoris cell culture medium.
Figura 9: Capacidad de la molécula trimérica H2V-DTetr para interferir con el efecto estimulador de la molécula hVEGFm.  Figure 9: Capacity of the trimeric molecule H2V-DTetr to interfere with the stimulatory effect of the hVEGFm molecule.
DIVULGACION DE LA INVENCIÓN DISCLOSURE OF THE INVENTION
La presente invención describe moléculas polipeptídicas recombinantes relacionadas con anticuerpos, que reconocen específicamente el Factor de Crecimiento de Endotelío Vascular-A (VEGF-A) humano, e interfieren con sus efectos estimulantes in vitro, y pro-angiogénicos in vivo.  The present invention describes recombinant polypeptide molecules related to antibodies, which specifically recognize the Human Vascular Endothelium Growth Factor (VEGF-A), and interfere with its stimulatory effects in vitro, and pro-angiogenic in vivo.
Estas moléculas comprenden las regiones variables de ¡nmunoglobulinas humanas que reconocen epítopes del VEGF-A humano y bloquean el efecto pro- angiogénico de esta molécula. Estas moléculas polipeptídicas están formadas por varios sitios, compuestos por aminoácidos codificados por secuencias de ADN de regiones variables de anticuerpos, de unión al VEFG-A. Las moléculas pueden ser anticuerpos recombinantes, fragmentos de anticuerpos de simple cadena (scFv), fragmentos de anticuerpos tipo Fab, o moléculas multiméricas. Dichas moléculas polipeptídicas recombinantes son capaces de inhibir la proliferación inducida por VEGF de células endoteliales humanas en ensayos in vitro. Estas moléculas pueden formar trímeros al fusionarse al dominio de trimerización de la tetranectina humana y pueden emplearse para la inmunoterapia pasiva de entidades patológicas cuyo curso se asocia al aumento de la vasculatura, tales como la degeneración macular asociada a la edad (variante húmeda), el cáncer y sus metástasis, los glaucomas neovasculares, la retinopatía diabética y del recién nacido, los procesos inflamatorios agudos y crónicos, enfermedades infecciosas, enfermedades autoinmunes, el rechazo al transplante de órganos, los hemangiomas y los angiofibromas, entre otros. These molecules comprise the variable regions of human immunoglobulins that recognize human VEGF-A epitopes and block the pro-angiogenic effect of this molecule. These polypeptide molecules are formed by several sites, composed of amino acids encoded by DNA sequences of variable regions of antibodies, binding to VEFG-A. The molecules can be recombinant antibodies, single chain antibody fragments (scFv), Fab type antibody fragments, or multimeric molecules. Such recombinant polypeptide molecules are capable of inhibiting VEGF-induced proliferation of human endothelial cells in in vitro assays. These molecules can form trimers when fused to the trimerization domain of human tetranectin and can be used for passive immunotherapy of pathological entities whose course is associated with increased vasculature, such as age-related macular degeneration (wet variant), the cancer and its metastases, neovascular glaucoma, diabetic and newborn retinopathy, acute and chronic inflammatory processes, infectious diseases, autoimmune diseases, organ transplant rejection, hemangiomas and angiofibromas, among others.
Estas moléculas polipeptídicas están fusionadas al dominio de trimerización de la tetranectina humana o cualquiera de las secuencias pertenecientes a la familia de lectinas tipo C, lo que le permite incrementar su tiempo de vida media en circulación.  These polypeptide molecules are fused to the trimerization domain of human tetranectin or any of the sequences belonging to the family of type C lectins, which allows it to increase its half-life in circulation.
La presente invención describe un nuevo enfoque para incrementar el tiempo de vida media en circulación de proteínas recombinantes de valor terapéutico.  The present invention describes a new approach to increase the half-life in circulation of recombinant proteins of therapeutic value.
La invención también describe moléculas multiméricas compuestas al menos por dos construcciones monoméricas de polipéptidos y además describe los métodos de preparación de estas construcciones génicas y polipéptidos. Estas moléculas multiméricas son para el uso terapéutico, como por ejemplo tratamiento de tumores mediante la inhibición de las propiedades pro-angiogénicas del VEGF o cualquier otra terapia utilizando polipéptidos relacionados con moléculas de anticuerpos.  The invention also describes multimeric molecules composed of at least two monomeric polypeptide constructs and further describes the methods of preparing these gene constructs and polypeptides. These multimeric molecules are for therapeutic use, such as tumor treatment by inhibiting the pro-angiogenic properties of VEGF or any other therapy using polypeptides related to antibody molecules.
Esta invención describe cualquier combinación de moléculas polipeptídicas relacionadas con anticuerpos, no necesariamente con reconocimiento por un mismo epítopo, con el dominio de trimerización de la tetranectina humana.  This invention describes any combination of antibody-related polypeptide molecules, not necessarily with recognition by the same epitope, with the trimerization domain of human tetranectin.
A los efectos de esta invención se define la siguiente terminología:  For the purposes of this invention the following terminology is defined:
· Anticuerpos recombinantes:  · Recombinant antibodies:
Describe polipéptidos derivados de inmunoglobulinas producidas parcial o totalmente de forma sintética (por la vía del ácido desoxirribonucleico (ADN) recombinante o de síntesis artificial de genes) con reconocimiento específico de un antígeno mediante uno o más dominios que interactúan con este, formados por combinaciones particulares de regiones variables de cadenas pesada y ligera de inmunoglobulinas, y que se denomina comúnmente sitio de unión al antígeno. Ejemplos de anticuerpos recombinantes son los llamados fragmentos de anticuerpos de simple cadena (scFv) obtenidos por ingeniería genética que comprenden uno o más sitios de unión al antígeno. En estos scFv los dominios VH y VL de un único anticuerpo se unen en diferente secuencia (VH-VL o VL-VH) con un segmento de unión peptídico (linker) que permite a los dos dominios asociarse para formar un sitio de unión al antígeno. Entre los fragmentos de anticuerpos recombinantes también podemos encontrar los dominios de anticuerpos (dAb) los cuales están formados por una sola región variable con uno o más sitios de unión por el antígeno. Estos dominios de anticuerpos son las moléculas derivadas de anticuerpos más pequeñas y su estructura presenta una gran similitud estructural con las regiones variables de los anticuerpos de llamas y tiburones. Describes polypeptides derived from immunoglobulins partially or totally synthetically produced (via recombinant deoxyribonucleic acid (DNA) or artificial gene synthesis) with specific recognition of an antigen through one or more domains that interact with it, formed by particular combinations of variable regions of heavy and light chains of immunoglobulins, and which is commonly referred to as the antigen binding site. Examples of recombinant antibodies are the so-called single chain antibody (scFv) fragments obtained by genetic engineering comprising one or more antigen binding sites. In these scFv the VH and VL domains of a single antibody bind in different sequence (VH-VL or VL-VH) with a peptide binding segment (linker) that allows the two domains to associate to form an antigen binding site . Among the recombinant antibody fragments we can also find the antibody domains (dAb) which are formed by a single variable region with one or more antigen binding sites. These antibody domains are the molecules derived from smaller antibodies and their structure exhibits great structural similarity with the variable regions of the flame and shark antibodies.
Los fragmentos de anticuerpos se pueden obtener a partir de bibliotecas de anticuerpos, donde un repertorio amplio de genes (ya sea sintéticos, semi-sintéticos o naturales) de las regiones variables de una especie se combinan al azar para producir asociaciones particulares de regiones variables de anticuerpos, que se exponen luego en la superficie de fagos filamentosos.  Antibody fragments can be obtained from antibody libraries, where a broad repertoire of genes (whether synthetic, semi-synthetic or natural) from the variable regions of a species are randomly combined to produce particular associations of variable regions of antibodies, which are then exposed on the surface of filamentous phages.
• Sitio de unión al antígeno.  • Antigen binding site.
El primer término describe la parte de un anticuerpo que interactúa específicamente con un antígeno (o parte de él). Cuando el antígeno es grande, un anticuerpo puede unirse sólo a una parte particular del antígeno, cuya parte se denomina epítopo. Un sitio de unión de un anticuerpo está formado principalmente por dos regiones variables de anticuerpo, La región variable de cadena ligera y la región variable de cadena pesada. El sitio de unión de anticuerpo se forma por la interacción no covalente de las regiones variables. El sitio de unión de un anticuerpo puede ser estabilizado de forma artificial mediante la unión de las dos regiones variables con un péptido de unión (linker) que no interfiera con sus propiedades de reconocimiento específico del antígeno. Este es el caso de un fragmento tipo scFv.  The first term describes the part of an antibody that interacts specifically with an antigen (or part of it). When the antigen is large, an antibody can bind only to a particular part of the antigen, whose part is called an epitope. An antibody binding site is primarily formed by two antibody variable regions, the light chain variable region and the heavy chain variable region. The antibody binding site is formed by the non-covalent interaction of the variable regions. The binding site of an antibody can be artificially stabilized by binding the two variable regions with a binding peptide (linker) that does not interfere with its antigen-specific recognition properties. This is the case of a scFv type fragment.
• Epítopo:  • Epitope:
El epítopo reconocido por el sitio de unión de un anticuerpo, en el caso de ser el antígeno una proteína, puede estar formado por una secuencia lineal de aminoácidos, o ser conformacional, entendiéndose por ello que los aminoácidos reconocidos por el sitio de unión del anticuerpo están cercanos en la estructura terciaria de la proteína, pero no son necesariamente secuenciales en su estructura primaria. En el caso de proteínas, el epítopo es por naturaleza una zona discreta, definida por un grupo particular de aminoácidos que interactúan con los del anticuerpo mediante enlaces no covalentes.The epitope recognized by the binding site of an antibody, in the case of being the antigen a protein, can be formed by a linear sequence of amino acids, or be conformational, it being understood that the amino acids recognized by the binding site of the antibody they are close in the tertiary structure of the protein, but not They are necessarily sequential in their primary structure. In the case of proteins, the epitope is by nature a discrete zone, defined by a particular group of amino acids that interact with those of the antibody through non-covalent bonds.
• Específico: • Specific:
Se refiere a la situación en la cual un anticuerpo o fragmento de este no presenta una unión significativa a otras moléculas diferentes de su pareja de unión específica. Este término es también aplicable al caso donde un sitio de unión al antígeno es específico para un epítopo particular que aparece en un número de antígenos relacionados o no, en cuyo caso el sitio de unión será capaz de unirse a varios antígenos que porten el mencionado epítopo.  It refers to the situation in which an antibody or fragment thereof does not exhibit significant binding to molecules other than its specific binding partner. This term is also applicable to the case where an antigen binding site is specific for a particular epitope that appears in a number of related or unrelated antigens, in which case the binding site will be able to bind to several antigens that carry said epitope. .
• Secuencia espadadora:  • Sequence spacer:
Se refiere a la secuencia polipeptídica formada por residuos de Serina y Glicina, que se incluyenentre fragmentos de anticuerpos y el dominio de trimerización para conferirle felxibilidad a la molécula de fusión (SEQID8).  It refers to the polypeptide sequence formed by Serine and Glycine residues, which is included between antibody fragments and the trimerization domain to confer felxibility to the fusion molecule (SEQID8).
· Dominio de trimerización (DTetr):  Trimerization domain (DTetr):
Se refiere a la región polipeptídica de la molécula de la tetranectina humana o cualquier integrante de la familia de lectinas tipo C descritas, cualquiera sea su origen. En una realización preferida de la invención se utiliza como dominio de trimerización a la región comprendida entre los aminoácidos 47 y 73 de la cadena A de la tetranectina humana (NCBI: CAA45860). La secuencia comprende un fragmento con una estructura de alfa hélice y la siguiente secuencia de aminoácidos: LKS LDTLSQEVALLKEQQALQTVCLK) (SEQID7)  It refers to the polypeptide region of the human tetranectin molecule or any member of the family of type C lectins described, whatever their origin. In a preferred embodiment of the invention, the region between amino acids 47 and 73 of the human tetranectin A chain (NCBI: CAA45860) is used as the trimerization domain. The sequence comprises a fragment with an alpha helix structure and the following amino acid sequence: LKS LDTLSQEVALLKEQQALQTVCLK) (SEQID7)
Los fragmentos de anticuerpos descritos en esta invención reconocen específicamente a las isotermas 121, 148, 165 y 189 del VEGF humano. Los fragmentos de anticuerpos tipo scFv son: H2V (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), y DI 1 VII (SEQID6), estos fueron seleccionados a partir de una biblioteca semisintética de scFv desplegados en fagos filamentosos.  The antibody fragments described in this invention specifically recognize isotherms 121, 148, 165 and 189 of human VEGF. The scFv-like antibody fragments are: H2V (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), and DI 1 VII (SEQID6), these were selected from one semi-synthetic scFv library deployed in filamentous phages.
Otro tipo de molécula es el fragmento de anticuerpo que está compuesto por las secuencias H2V,(SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), y D11VII(SEQID6) fusionados al dominio de trimerización de la tetranectina humana. (SEQID7) y separados por una secuencia espadadora (SEQID8). Los efectos anti-anti-angiogénicos que se obtienen mediante la aplicación de las moléculas descritas en esta invención se producen debido a que interfieren con la interacción del VEGF-A humano con los receptores presentes en las células endoteliales vasculares activadas, lo que influye en la capacidad de estas para proliferar y mantener su estabilidad fisiológica. Another type of molecule is the antibody fragment that is composed of the sequences H2V, (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), and D11VII (SEQID6) fused to trimerization domain of human tetranectin. (SEQID7) and separated by a spacer sequence (SEQID8). The anti-anti-angiogenic effects that are obtained by applying the molecules described in this invention occur because they interfere with the interaction of human VEGF-A with the receptors present in the activated vascular endothelial cells, which influences the their ability to proliferate and maintain their physiological stability.
Además, pueden estar en la forma de moléculas de anticuerpo bi-específicos, donde una porción de las mismas conserven su especificidad para el VEGF-A humano (o cualquier antígeno) y otra tenga una especificidad diferente. Todas estas manipulaciones por ingeniería genética son conocidas para aquellos expertos en el arte.  In addition, they may be in the form of bi-specific antibody molecules, where a portion thereof retains its specificity for human VEGF-A (or any antigen) and another has a different specificity. All these manipulations by genetic engineering are known to those skilled in the art.
Secuencias: Sequences:
SEQID1.H2V SEQID1.H2V
MAEVQLLESGGGLVQPGGSLRLSCASSSSSSSSSSMSWVRQAPGKGLEWYSYYYSYSYGYTGYA DSV GRFnSADTSKNTAYLQMNSLRAEDTAVYVECKNGANDDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG AP LLIYSASFLY SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCSYYSYSSYTFGQGTKVEIK  MAEVQLLESGGGLVQPGGSLRLSCASSSSSSSSSSMSWVRQAPGKGLEWYSYYYSYSYGYTGYA DSV GRFnSADTSKNTAYLQMNSLRAEDTAVYVECKNGANDDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ PG AP LLIYSASFLY SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCSYYSYSSYTFGQGTKVEIK
SEQID2.B5VIII  SEQID2.B5VIII
MAEVQLLESGGGLVQPGGSLRLSCAYSSSSSYYYSMSWVRQAPGKGLEWSSSYSSSYSGYTGYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYQETCQTYNADYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCYSYYYYYYTFGQGTKVEIK  MAEVQLLESGGGLVQPGGSLRLSCAYSSSSSYYYSMSWVRQAPGKGLEWSSSYSSSYSGYTGYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYQETCQTYNADYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCYSYYYYYYTFGQGTKVEIK
SEQID3:H8V SEQID3: H8V
MAEVQLLESGGGLVQPGGSLRLSCASSSSSSYYSSMSWVRQAPGKGLEWSSSSSSYSSGYTGYA DSVKGRFnSADTSKNTAYLQMNSLRAEDTAVYTRYRNQDKTDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCYYYSYYYYTFGQGTKVEIK  MAEVQLLESGGGLVQPGGSLRLSCASSSSSSYYSSMSWVRQAPGKGLEWSSSSSSYSSGYTGYA DSVKGRFnSADTSKNTAYLQMNSLRAEDTAVYTRYRNQDKTDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCYYYSYYYYTFGQGTKVEIK
SEQID4:E8V SEQID4: E8V
AEVQLLESGGGLVQPGGSLRLSCASYSYYSSSSYMSWVRQAPGKGLEWSSYSYSSYSGYTGYA DSVKGRFnSADTSKNTAYLQMNSLRAEDTAVYSTSKTADNCDYWGQGTLVTVSSGGGGSGGG GSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTmSSLQPEDFATYYCYSYYYSYYTFGQGTKVEIK SEQID5.H6VII AEVQLLESGGGLVQPGGSLRLSCASYSYYSSSSYMSWVRQAPGKGLEWSSYSYSSYSGYTGYA DSVKGRFnSADTSKNTAYLQMNSLRAEDTAVYSTSKTADNCDYWGQGTLVTVSSGGGGSGGG GSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTmSSLQPEDFATYYCYSYYYSYYTFGQGTKVEIK SEQID5.H6VII
MAEVQLLESGGGLVQPGGSLRLSCASSYYSSSSSSMSWVRQAPGKGLEWSYYSYSSSYGYTGYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYLSSNRASDSDYWGQGTLVTVSSGGGGSGGG GSGGGGSTDIQMTQSPSSLSASVGDRVT1TCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTmSSLQPEDFATYYCYYSSYSYSTFGQGTKVEIK MAEVQLLESGGGLVQPGGSLRLSCASSYYSSSSSSMSWVRQAPGKGLEWSYYSYSSSYGYTGYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYLSSNRASDSDYWGQGTLVTVSSGGGGSGGG GSGGGGSTDIQMTQSPSSLSASVGDRVT1TCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTmSSLQPEDFATYYCYYSSYSYSTFGQGTKVEIK
SEQID6:D11VII AEVQLLESGGGLVQPGGSLRLSCASYYYSSYSYSMSWVRQAPG GLEWYSSSSYYSYGYTGYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYPCNYETRESDYWGQGTLVTVSSGGGGSGGG GSGGGGSTDIQMTQSPSSLSASVGDRVnTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTmSSLQPEDFATYYCYSYYYSSYTFGQGTKVEIK  SEQID6: D11VII AEVQLLESGGGLVQPGGSLRLSCASYYYSSYSYSMSWVRQAPG GLEWYSSSSYYSYGYTGYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYPCNYETRESDYWGQGTLVTVSSGGGGSGGG GSGGGGSTDIQMTQSPSSLSASVGDRVnTCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTmSSLQPEDFATYYCYSYYYSSYTFGQGTKVEIK
SEQID7: Dominio tetranectina aa 47-73 cadena A. SEQID7: Tetranectin domain aa 47-73 chain A.
L SRLDTLSQEVALLKEQQALQTVCLK SEQID8. Péptido espaciador.  L SRLDTLSQEVALLKEQQALQTVCLK SEQID8. Peptide spacer.
SGGGG  SGGGG
EJEMPLOS DE APLICACIÓN. APPLICATION EXAMPLES
Ejemplo 1: Clonaje y expresión en células SiHa y purificación de la isoforma 121 del VEGF.  Example 1: Cloning and expression in SiHa cells and purification of isoform 121 of VEGF.
a) Clonaje de la Isoforma 121 del VEGF humano.  a) Cloning of Isoform 121 of human VEGF.
La secuencia del VEGF 121 humano se obtuvo de la bases de datos del NCBI (GenBank: AAF19659.1). Por ser la de menor tamaño, todos los epítopos presentes en esta variante, también están contenidos en las otras isoformas de VEGF humano. De esta forma se garantiza que los anticuerpos, generados contra la variante 121, también serán capaces de reconocer las variantes de VEGF 189 y 206. Con el propósito de facilitar su posterior detección y purificación se decidió incorporar una cola de 6 residuos de histidina (6HIS) en uno de los extremos de la molécula de hVEGFm. The human VEGF 121 sequence was obtained from the NCBI databases (GenBank: AAF19659.1). Being the smallest, all epitopes present in this variant, are also contained in the other isoforms of human VEGF. This ensures that the antibodies, generated against variant 121, will also be able to recognize variants of VEGF 189 and 206. In order to facilitate their subsequent detection and purification, it was decided to incorporate a tail of 6 histidine residues (6HIS ) at one end of the hVEGFm molecule.
El gen codificante para la variante del hVEGFm, fue sintetizado por la empresa norteamericana Blu Heron. Para facilitar los posteriores pasos de clonamiento, el gen de hVEGFm fue flanqueado por los sitios de restricción Xho I y EcoR V. Mediante la digestión con estas enzimas se extrajo la banda correspondiente al gen hVEGF 121 y se clonó en el vector de transferencia adenoviral pAdTrack-CMV, perteneciente al kit "AdEasyTM Adenoviral Vector System", de Agilent Technologies. Como resultado de este clonamiento se obtuvo el vectorpAdTrack- hVEGFm (Figura 1), el cual se extrajo mediante la digestión enzimática con las endonucleasas Xho I y EcoRV para ser insertado en el vector adenoviral previamente digerido con estas endonucleasas. Con este vector se obtuvieron partículas adenovirales en células HEK-293. Obteniéndose títulos virales de 2,44 xlO10 unidades formadoras de colonias por mL (UFC/mL) The gene coding for the hVEGFm variant was synthesized by the North American company Blu Heron. To facilitate subsequent cloning steps, the hVEGFm gene was flanked by restriction sites Xho I and EcoR V. By digestion with these enzymes, the band corresponding to the hVEGF 121 gene was extracted and cloned into the adenoviral transfer vector pAdTrack -CMV, belonging to the "AdEasyTM kit Adenoviral Vector System ", by Agilent Technologies. As a result of this cloning the vectorpAdTrack-hVEGFm (Figure 1) was obtained, which was extracted by enzymatic digestion with the endonucleases Xho I and EcoRV to be inserted into the adenoviral vector previously digested with these endonucleases With this vector, adenoviral particles were obtained in HEK-293 cells, obtaining viral titers of 2.44 x 10 10 colony forming units per mL (CFU / mL)
b) Expresión y purificación de la proteína en células SIHa.  b) Expression and purification of the protein in SIHa cells.
Utilizando una MOI (multiplicidad de infección viral) de 80 se infectaron células Sil-la para la expresión de hVEGFm. Después de la cosecha, el medio de cultivo fue sometido a una cromatografía de afinidad de iones metálicos usando como matriz la Chelating Sepharose Fast Flow. La purificación del sobrenadante de cultivo se realizó mediante cromatografía de afinidad de quelatos metálicos. La figura 2A muestra el cromatograma del proceso. La figura 2B muestra la electroforesis en geles de pliacrilamida al 12,5 % donde se observan de izquierda a derecha: medio de entrada, proteínas no unidas, lavado con 80 mM de imidaziol, y elución con 250 mM de imidazol. Para la cromatografía se adicionó imidazol al medio de cultivo hasta alcanzar una concentración de 5 mM. La matriz fue lavada con 80 mM de imidazol en buffer fosfato pH 7,2, y se realizó un simple paso de elución en 250 mM de imidazol. Se analizaron las fracciones mediante electroforesis, en condiciones reductoras, de cada una de las etapas de la cromatografía. El proceso de purificación rindió dos bandas dentro del rango de 15 a 20 kDa que parecen ser las formas glicosiladas y no glicosilasas del hVEGFm. Using an MOI (multiplicity of viral infection) of 80 Sil-la cells were infected for hVEGFm expression. After harvesting, the culture medium was subjected to metal ion affinity chromatography using the Chelating Sepharose Fast Flow as a matrix. Purification of the culture supernatant was performed by affinity chromatography of metal chelates. Figure 2A shows the process chromatogram. Figure 2B shows electrophoresis in 12.5% pliacrylamide gels where they are observed from left to right: entry medium, unbound proteins, washed with 80 mM imidaziol, and elution with 250 mM imidazole. For chromatography, imidazole was added to the culture medium until it reached a concentration of 5 mM. The matrix was washed with 80 mM imidazole in phosphate buffer pH 7.2, and a simple elution step was performed in 250 mM imidazole. The fractions were analyzed by electrophoresis, under reducing conditions, of each of the chromatography stages. The purification process yielded two bands within the range of 15 to 20 kDa that appear to be the glycosylated and non-glycosylase forms of hVEGFm.
c) Ensayo de actividad proliferativa del VEGF purificado.  c) Proliferative activity test of purified VEGF.
Se determinó la actividad proliferativa "t'n sitW e nfn vivó' del hVEGFm producido en el laboratorio. The proliferative activity "t ' n sitW e n fn viv' of the hVEGFm produced in the laboratory was determined.
Para determinar la actividad n/n sitW se realizó un cultivo primario de células HUVEC (pase 4). Las células fueron cultivadas en placas de 96 pocilios a razón de 10000 células por pocilio. Se evaluó la actividad de hVEGF121 en el rango de concentración entre 0,1 y 1000 ng/ml. Pasadas 72 horas de cultivo, las células fueron teñidas con MTT (3-(4, 5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), y la absorbancia se leyó a 570 nm. Los resultados son mostrados como la media + SD de tres pocilios. (A) 10 000 células por well a momento de la siembra. (B) 20000 células por pocilio a momento de la siembra (Figura 3). La actividad de proangiogénica del hVEGF121 se determinó mediante un ensayo de angiogénesis n/n vivd' usando matrigel (BD). Se hizo un grupo experimental donde la mezcla de matrigel contenía heparina (64 UI/mL) y hVEGFm (30 ng/mL). De esta mezcla se inyectaron 300 μΙ_ por vía subcutánea en tres ratones híbridos CFl-BALB/c. Como control negativo se emplearon tres ratones a los que se les inyectó una mezcla de matrigel y heparina, carente de hVEGFm. Siete días después de la inyección, los ratones fueron sacrificados y se les extrajo tapón de matrigel. En la figura 4 se presenta estos resultados ratones inoculados con matrigel, con (A) o sin hVEGF 121 (B) purificado (30 ng/mL). La figura 4C muestra la cuantificación de la neovascularización en los tapones de matrigel mediante la medición del contenido de hemoglobina. To determine the n / n sitW activity, a primary culture of HUVEC cells was performed (step 4). The cells were cultured in 96-well plates at a rate of 10,000 cells per well. The activity of hVEGF121 in the concentration range between 0.1 and 1000 ng / ml was evaluated. After 72 hours of culture, the cells were stained with MTT (3- (4, 5- dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide), and the absorbance was read at 570 nm. The results are shown as the mean + SD of three wells. (A) 10,000 cells per well at planting time. (B) 20,000 cells per well at planting time (Figure 3). The proangiogenic activity of hVEGF 121 was determined by an n / n vivd 'angiogenesis assay using matrigel (BD). An experimental group was made where the matrigel mixture contained heparin (64 IU / mL) and hVEGFm (30 ng / mL). Of this mixture, 300 μΙ_ were injected subcutaneously into three CFL-BALB / c hybrid mice. As a negative control, three mice were used to inject a mixture of matrigel and heparin, lacking hVEGFm. Seven days after the injection, the mice were sacrificed and a matrigel plug was removed. Figure 4 shows these results mice inoculated with matrigel, with (A) or without purified hVEGF 121 (B) (30 ng / mL). Figure 4C shows the quantification of neovascularization in matrigel plugs by measuring hemoglobin content.
La flecha muestra la formación de vasos sanguíneos en el interior del tapón de matrigel. (B) Ratón inoculado matrigel carente hVEGF121. No se observan vasos en el interior de tapón. (C) la neovascularización en los tapones de matrigel se cuantificó mediante medición del contenido de hemoglobina en el tapón con el método de Drabkin, los resultados son mostrados como la media ± SD dé tres tapones. Los resultados obtenidos sugieren que el hVEGF producido por esta metodología es biológicamente activo.  The arrow shows the formation of blood vessels inside the matrigel plug. (B) Matrix inoculated mouse lacking hVEGF121. No glasses are observed inside the cap. (C) the neovascularization in the matrigel caps was quantified by measuring the hemoglobin content in the cap with the Drabkin method, the results are shown as the mean ± SD of three caps. The results obtained suggest that the hVEGF produced by this methodology is biologically active.
Ejemplo 2: Selección de fragmentos de anticuerpos de alta afinidad contra el VEGF humano. Example 2: Selection of high affinity antibody fragments against human VEGF.
Para la selección de fragmentos de anticuerpos contra el VEGF humano se utilizó una biblioteca semi-sintética de fragmentos de anticuerpos de simple cadena (scFv), sobre fagos filamentosos construida especialmente para esta invención. En ella se aleatorizaron, las regiones determinantes de la complementariedad (CDR) (L3, Hl, H2 y H3) de las cadenas variables del fragmento de anticuerpos FD5 (PDB ID IFVC) como andamio proteico.  For the selection of antibody fragments against human VEGF, a semi-synthetic library of single chain antibody fragments (scFv) was used, on filamentous phages specially constructed for this invention. In it, the complementarity determining regions (CDR) (L3, Hl, H2 and H3) of the variable chains of the FD5 antibody fragment (PDB ID IFVC) as protein scaffold were randomized.
En las selecciones a partir de esta biblioteca se empleó como antígeno la proteína hVEGFi2i humano portando la etiqueta 6HIS. Se utilizaron placas recubiertas con Níquel para capturar la etiqueta 6HIS de la cual es portadora esta variante de VEGF humano. In the selections from this library, the human hVEGFi 2 i protein carrying the 6HIS tag was used as an antigen. Nickel-coated plates were used to capture the 6HIS tag of which this human VEGF variant is a carrier.
Se recubrieron pocilios de estas placas con 5 ug/mL de la proteína en PBS, a 4°C durante toda la noche y luego se bloquearon con PBS-Leche descremada 4%. Los fagos que no se unieron se eliminaron mediante 20 lavados con una solución de PBS-Tween al 0,1 %, seguido por 4 lavados con solución glicina-HCL (0,2 M, pH 2,2). A continuación, los fagos unidos se eluyeron con solución glicina-HCL (0,2 M, pH 2,2) y sonicación 50 kHz durante 10 minutos en baño con agua. La fracción eluída se neutralizó con solución tampón fosfato (200 mM, pH 7,5). Esta variante de elución permitirá obtener ligandos de alta afinidad28. Wells of these plates were coated with 5 ug / mL of the protein in PBS, at 4 ° C overnight and then blocked with PBS-Skimmed Milk 4%. Phage that did not bind were removed by 20 washes with a 0.1% PBS-Tween solution, followed by 4 washes with glycine-HCL solution (0.2 M, pH 2.2). Then, the bound phages were eluted with glycine-HCL solution (0.2 M, pH 2.2) and 50 kHz sonication for 10 minutes in a water bath. The eluted fraction was neutralized with phosphate buffer solution (200 mM, pH 7.5). This elution variant will allow obtaining high affinity ligands 28 .
Los fagos eluídos se amplificaron en la cepa TG 1 de £ cofí se usaron como material de partida en el próximo ciclo de selección. Este procedimiento se repitió 3 veces bajo las mismas condiciones. Posteriormente, se seleccionaron al azar colonias individuales de TG1 infectadas con los fagos eluídos del segundo y tercer ciclo de selección para producir fagos a escala de 96 pozos. La capacidad de estos clones de fagos que portan fragmentos scFv de anticuerpo para unir VEGF se evaluó mediante ELISA. Para ello se recubrieron placas de 96 pozos Maxisorp con 5 pg/mL de VEGF, y luego se bloquearon. Los fagos diluidos en PBS-leche descremada al 4% se incubaron durante 1 hora a 25°C en las placas, seguido por varios lavados. Los fagos unidos se detectaron mediante la adición de anticuerpos anti-M13 conjugados a peroxidasa (Amersham) por 1 hora a 25°C. Luego de varios lavados, la reacción se reveló con la adición de solución sustrato de la peroxidasa (TMB). La absorbancia se leyó a 450 nm en un lector de microplacas. De los 90 clones evaluados 57 resultaron positivos.  The eluted phages were amplified in the TG1 strain of the cofi were used as starting material in the next selection cycle. This procedure was repeated 3 times under the same conditions. Subsequently, individual colonies of TG1 infected with the eluted phages of the second and third selection cycle were randomly selected to produce phages at 96-well scale. The ability of these phage clones that carry scFv antibody fragments to bind VEGF was evaluated by ELISA. For this, Maxisorp 96-well plates were coated with 5 pg / mL of VEGF, and then blocked. The phages diluted in PBS-4% skim milk were incubated for 1 hour at 25 ° C in the plates, followed by several washes. Bound phages were detected by the addition of peroxidase-conjugated anti-M13 antibodies (Amersham) for 1 hour at 25 ° C. After several washes, the reaction was developed with the addition of peroxidase substrate solution (TMB). The absorbance was read at 450 nm in a microplate reader. Of the 90 clones evaluated, 57 were positive.
Ejemplo 3: Selección de los clones mediante la estimación de sus afinidades por el VEGF. Example 3: Selection of clones by estimating their affinities for VEGF.
La estimación de las afinidades se realizó mediante un ensayo ELISA competitivo de un solo punto. Los fagos diluidos en PBS-Tween con o sin 100 nM VEGF se incubaron durante 1 hora a 25°C. Las mezclas fueron transferidas a placas de ELISA Maxisorp (Nunc) recubiertas con VEGF e incubadas durante 15 minutos a la misma temperatura, seguido de varios lavados. Los fagos unidos se detectaron mediante la adición de anticuerpos anti-M13 conjugados a peroxidasa (Amersham) por 1 hora a 25°C. Luego de varios lavados, la reacción se reveló con la adición de solución sustrato de la peroxidasa (TMB). La absorbancia se leyó a 450 nm en un lector de microplacas.  The affinities were estimated using a single-point competitive ELISA. The phages diluted in PBS-Tween with or without 100 nM VEGF were incubated for 1 hour at 25 ° C. The mixtures were transferred to Maxisorp ELISA plates (Nunc) coated with VEGF and incubated for 15 minutes at the same temperature, followed by several washes. Bound phages were detected by the addition of peroxidase-conjugated anti-M13 antibodies (Amersham) for 1 hour at 25 ° C. After several washes, the reaction was developed with the addition of peroxidase substrate solution (TMB). The absorbance was read at 450 nm in a microplate reader.
Se seleccionaron 6 clones H2V (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), y D11VII (SEQID6). El gráfico (Figura 5) muestra los resultados basados en la afinidad relativa de los clones seleccionados. Los fagos portadores de fragmentos scFv que reconocen el hVEGF121 se incubaron con ΙΟΟηΜ de hVEGF121 y sin hVEGF121, luego se añadieron a una placa recubierta con hVEGF121. Se seleccionaron los que mostraron menores valores de la relación de absorbancias de la condición con VhEGF121 y sin hVEGF121. 6 clones H2V (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), and D11VII (SEQID6) were selected. The graph (Figure 5) shows the results based on the relative affinity of the selected clones. Phages carriers of scFv fragments that recognize hVEGF121 were incubated with ΙΟΟηΜ of hVEGF121 and without hVEGF121, then added to a plate coated with hVEGF121. Those that showed lower values of the absorbance ratio of the condition with VhEGF121 and without hVEGF121 were selected.
Ejemplo 4: Clonaje de fragmento scFv H2V-DTetr en el vector pPSIO. Example 4: Cloning of scFv H2V-DTetr fragment in the pPSIO vector.
El vector pPSIO es un plasmidio diseñado para la expresión en la levadura P. Pastoris. Este vector contienes como elementos principales el origen de replicación y el gen de la β-lactamasa de E col/, un fragmento regulador del promotor del gen AOX1 de P. pastoris, la señal de secreción de la enzima invertasa de la sacarosa (ssSUC2), codificada por el gen SUC2 de S. cerevisiae, el terminador de la transcripción del gen de la gliceraldehído 3 fosfato deshidrogenasa (tGAP) de S. cerevisiae, el gen HIS3 de 5. cerevisiae y la región 3' del gen AOX1 de P. pastoris (Figura 6).  The pPSIO vector is a plasmid designed for expression in P. Pastoris yeast. This vector contains as main elements the origin of replication and the gene of β-lactamase of E col /, a regulatory fragment of the promoter of the AOX1 gene of P. pastoris, the secretion signal of the sucrose invertase enzyme (ssSUC2) , encoded by the S. cerevisiae SUC2 gene, the transcription terminator of the S. cerevisiae glyceraldehyde 3 phosphate dehydrogenase (tGAP) gene, the 5. cerevisiae HIS3 gene and the 3 'region of the AOX1 gene of P. pastoris (Figure 6).
A continuación se describe el diseño realizado de la molécula scFvH2V-DTetr (Tabla 1). Esta molécula contiene la secuencia de amino ácidos del clon H2V del fragmento de anticuerpo tipo scFv que reconoce la molécula de VEGF humana isoforma 121. Este fragmento se encuentra unido a través una secuencia peptídica al dominio de trimerización de la tetranectina humana. (SEQID7). Esta secuencia, que abarca desde el amino ácido 42 al 73 de la tetranectina humana de la cadena A, es la que permite formar los trímeros de scFvH2V-DTetr.  The design of the scFvH2V-DTetr molecule is described below (Table 1). This molecule contains the amino acid sequence of the H2V clone of the scFv-like antibody fragment that recognizes the human VEGF molecule isoform 121. This fragment is linked through a peptide sequence to the trimerization domain of human tetranectin. (SEQID7). This sequence, which ranges from amino acid 42 to 73 of the human tetranectin of the A chain, is what makes it possible to form the scFvH2V-DTetr trimers.
Tabla 1: Secuencia de la molécula scFvH2V-DTetr.  Table 1: Sequence of the scFvH2V-DTetr molecule.
MAEVQLLESGGGLVQPGGSLRLSCASSSSSSSSSSMSWV QAPGKGLEWYSYYYSYSYGYTGYA DSVKGRmSADTSKNTAYLQMNSLRAEDTAVWECKNGANDDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTYVAWYQQKPGKAPKLLIYSASFLY I SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCSYYYYYSYTFGQGTKVEIKSGGGGLKSRLDTLS QEVALLKEQQALQTVCLK MAEVQLLESGGGLVQPGGSLRLSCASSSSSSSSSSMSWV QAPGKGLEWYSYYYSYSYGYTGYA DSVKGRmSADTSKNTAYLQMNSLRAEDTAVWECKNGANDDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTTTCRASQDVNTYVAWYQQKPGKAPKLLIYSASFLY I SGVPSRFSGSRSGTDFTmSSLQPEDFATYYCSYYYYYSYTFGQGTKVEIKSGGGGLKSRLDTLS QEVALLKEQQALQTVCLK
La secuencia codificantes correspondiente al clon H2V fusionado al dominio de trimerización de la tetranectina humana (DTetr) (SEQID7) se obtuvieron por síntesis química a la cual se le añadieron los sitios de reconocimiento para las endonucleasas de restricción Smal y Nael. Este gen fue proporcionado por la compañía GenScript en el plasmidio comercial pUC-K (Figura 6). El dominio de trimerización de la tetranectina conformados por la secuencia de amino ácidos (43-73) de la tetranectina humana permite la formación de la molécula trimérica formada por monómeros de la proteína de fusión scFv H2V-DTetr (Figura 7) The coding sequence corresponding to the H2V clone fused to the trimerization domain of human tetranectin (DTetr) (SEQID7) was obtained by chemical synthesis to which the recognition sites for Smal and Nael restriction endonucleases were added. This gene was provided by the GenScript company in the commercial plasmid pUC-K (Figure 6). The trimerization domain of tetranectin formed by the amino acid sequence (43-73) of human tetranectin allows the formation of the trimeric molecule formed by scFv H2V-DTetr fusion protein monomers (Figure 7)
El vector pPSIO fue digerido con las enzimas de restricción Smal y Nael Los extremos del vector lineal se desfosforilaron posteriormente con fosfatasa antártica. El plásmidio que contenía el fragmento del scFv H2V-DTetr fue digerido con las enzimas Smal y Nael y se purificó junto al vector a partir de un gel de agarosa de bajo punto de fusión al 0,8%. Los fragmentos de interés se ligaron con la enzima ADN T4 ligasa, la reacción se transformó en células de E coi/y los plasmidios recombinantes se detectaron por ensayos de restricción con las mismas enzimas. El plásmido de expresión resultante se denominó pPscFvH2V-Dtetr.  The pPSIO vector was digested with the restriction enzymes Smal and Nael. The ends of the linear vector were subsequently dephosphorylated with Antarctic phosphatase. The plasmid containing the scFv H2V-DTetr fragment was digested with the enzymes Smal and Nael and purified next to the vector from a 0.8% low melting agarose gel. The fragments of interest were ligated with the enzyme T4 DNA ligase, the reaction was transformed into E coi / cells and the recombinant plasmids were detected by restriction assays with the same enzymes. The resulting expression plasmid was called pPscFvH2V-Dtetr.
Ejemplo 5: Transformación, expresión y purificación de la proteína multimérica pPscFvH2V-DTetr en Pichia pastoris. Example 5: Transformation, expression and purification of the multimeric protein pPscFvH2V-DTetr in Pichia pastoris.
a) Transfromación de la construcción pPscFvH2V en Pichia Pastoris.  a) Transfromation of the pPscFvH2V construct in Pichia Pastoris.
La transformación de células competentes de la cepa hospedante MP36 y el análisis de los transformantes se realizó según el procedimiento descrito por Martínez y colaboradores (1993). Se utilizaron 10 pg del plásmido de expresión, digeridos previamente con las enzimas Salí. Se utilizaron cubetas de 0,2 mm, en el equipo MicroPulser™ (Bio-Rad, EE.UU.) seleccionando el programa predeterminado "fungí". Después del pulso se añadió a las cubetas 1 mL de sorbitol 1 M. El producto de la transformación se sembró en placas de medio mínimo YNB-G (carente de histidina), a 28°C por 6 días.  The transformation of competent cells of the host strain MP36 and the analysis of the transformants was performed according to the procedure described by Martínez et al. (1993). 10 pg of the expression plasmid, previously digested with the Salí enzymes, were used. 0.2 mm cuvettes were used in the MicroPulser ™ device (Bio-Rad, USA) by selecting the default "fungí" program. After the pulse, 1 mL of 1 M sorbitol was added to the cuvettes. The transformation product was seeded in YNB-G minimum plates (lacking histidine), at 28 ° C for 6 days.
Después de extraer el ADN cromosomal de las colonias crecidas (Martínez y cois., 1993), las muestras se analizaron mediante hibridación de ácidos nucleicos por "Southern blot", para comprobar la integración de los casetes de expresión en el genoma de la levadura. Para este estudio, el ADN cromosomal aislado, correspondiente a cada transformante, se digirió con la enzima de restricción EcoRI. Se utilizaron dos sondas para el estudio de los transformantes; una de ellas consistió en el gen que codifica el fragmento de anticuerpo scFvH2V-DTetr obtenido por Digestión del plásmido pUC-K_scFvH2V-DTetr con Smal y Nael, y otra en un fragmento del promotor del gen AOX1. En las hibridaciones se empleó como control positivo el ADN del plásmido. Como control negativo en cada caso se utilizó el ADN de la cepa hospedera MP36 sin transformar. b) Expresión After extracting the chromosomal DNA from the grown colonies (Martínez et al., 1993), the samples were analyzed by nucleic acid hybridization by "Southern blot", to check the integration of the expression cassettes in the yeast genome. For this study, the isolated chromosomal DNA, corresponding to each transformant, was digested with the restriction enzyme EcoRI. Two probes were used for the study of transformants; one of them consisted of the gene encoding the scFvH2V-DTetr antibody fragment obtained by digestion of plasmid pUC-K_scFvH2V-DTetr with Smal and Nael, and another in a fragment of the AOX1 gene promoter. In the Hybridizations were used as positive control plasmid DNA. As a negative control in each case, the DNA of the untransformed MP36 host strain was used. b) Expression
Los experimentos de inducción se realizaron en matraces Erlenmeyer de 1 L de capacidad. Se partió con un pre-inoculo de 10 ml_ preparado en medio de cultivo YPG, este se cultivó durante el 20 horas en agitación orbital (240 rpm) a 28°C. A partir del pre-inoculo ya crecido, se inoculó un cultivo de 500 ml_ de medio YP suplementado con glicerol al 2%. Al cabo de 20 hrs el medio se centrifugó a 3.500 rpm por 5 min, se eliminó el sobrenadante y el pellet se resuspendió en 500mL de medio YP. La inducción de la expresión de la proteína scFvH2V-DTetr se comenzó inmediatamente suplementando el medio YP con Metanol al 1% cada 12 horas manteniendo el cultivo en formato batch a 28°C en agitación orbital (240 rpm). Se midió la densidad óptica (600 nm) del cultivo cada 24 horas para estudiar la cinética de crecimiento durante la inducción. Luego de 72 horas post-inducción se detuvo ia adición de metanol. Una vez finalizada la inducción las células fueron colectadas por centrifugación a 6200 rpm por 5 minutos y el sobrenadante de cultivo se almacenó a -20°C para su posterior clarificación. Induction experiments were performed in Erlenmeyer flasks of 1 L capacity. It was split with a pre-inoculum of 10 ml_ prepared in YPG culture medium, this was grown for 20 hours in orbital shaking (240 rpm) at 28 ° C. From the pre-inoculum already grown, a 500 ml culture of YP medium supplemented with 2% glycerol was inoculated. After 20 hrs the medium was centrifuged at 3,500 rpm for 5 min, the supernatant was removed and the pellet was resuspended in 500mL of YP medium. Induction of scFvH2V-DTetr protein expression was started immediately by supplementing the YP medium with 1% Methanol every 12 hours keeping the batch culture at 28 ° C in orbital shaking (240 rpm). The optical density (600 nm) of the culture was measured every 24 hours to study the growth kinetics during induction. After 72 hours post-induction, the addition of methanol was stopped. Once the induction was finished, the cells were collected by centrifugation at 6200 rpm for 5 minutes and the culture supernatant was stored at -20 ° C for later clarification.
c) Purificación de la proteína multimérica  c) Purification of the multimeric protein
Los 500mL de medio de cultivo se centrifugaron a 6200 rpm por 5 minutos, se recuperó el sobrenadante el que se centrifugó a 8.000 rpm por 15 minutos, el sobrenadante se clarificó a través de un pre-filtro de Fibra de vidrio y finalmente filtrado a 0.45 -m.  The 500mL of culture medium was centrifuged at 6200 rpm for 5 minutes, the supernatant was recovered which was centrifuged at 8,000 rpm for 15 minutes, the supernatant was clarified through a fiberglass pre-filter and finally filtered at 0.45 -m.
El medio clarificado se sometió a ultrafiltración en membrana de nitrocelulosa con un tamaño de exclusión de lOKDa. El medio se concentró y durante este proceso se remplazó con 150 mL de Tampón fosfato 50mM pH 7,0.  The clarified medium was subjected to ultrafiltration in nitrocellulose membrane with an exclusion size of lOKDa. The medium was concentrated and during this process it was replaced with 150 mL of 50mM phosphate buffer pH 7.0.
Se purificó un total de 15 mi de sobrenadante de cultivo clarificado y concentrado mediante cromatografía de afinidad por iones metálicos, para ello se utilizó la resina de quelatos TOYOPEARL (TOYO, Japón).  A total of 15 ml of clarified and concentrated culture supernatant was purified by metal ion affinity chromatography, for which the TOYOPEARL chelate resin (TOYO, Japan) was used.
La muestra de entrada se cargó a pH 8,0 con 5 mM de Imidazol, se dejó fluir por gravedad a temperatura ambiente, la unión inespecífica de proteínas fue eliminada mediante un paso de lavado con 50 mM de imidazol en tampón fosfato a pH 7,5, La elución final de la proteína se realizó a 250 mM de Imidazol en tampón fosfato pH7,0. Se colectaron las fracciones de entrada, muestra no unida, lavado y elución obtenidas del proceso de purificación. Estas fracciones se sometieron a una electroforesis de proteínas en geles de acrilamida y Westen-Blot en condiciones no desnaturalizantes. Los resultados de estos experimentos muestran la formación de la molécula trimérica scFvH2V-DTetr (Figura 8), donde PPM: Patrón de peso molecular, línea 1: medio de entrada, línea 2: proteínas no unidas, línea 3: lavado con 50 mM de imidaziol, línea 4: elución con 250 mM de imidazol. (B) Western-blot en condiciones no desnaturalizantes del proceso de purificación por cromatografía de afinidad por iones metálicos. PPM: Patrón de peso molecular, línea 1: medio de entrada, línea 2: proteínas no unidas, línea 3: lavado con 50 mM de imidaziol, línea 4: elución con 250 mM de imidazol. The inlet sample was loaded at pH 8.0 with 5 mM Imidazole, allowed to flow by gravity at room temperature, nonspecific protein binding was removed by a 50 mM imidazole wash step in phosphate buffer at pH 7, 5, Final elution of the protein was performed at 250 mM Imidazole in phosphate buffer pH7.0. The input fractions, unbound sample, washing and elution obtained were collected of the purification process. These fractions were subjected to protein electrophoresis in acrylamide and Westen-Blot gels under non-denaturing conditions. The results of these experiments show the formation of the scFvH2V-DTetr trimeric molecule (Figure 8), where PPM: Molecular weight pattern, line 1: entry medium, line 2: unbound proteins, line 3: wash with 50 mM of imidaziol, line 4: elution with 250 mM imidazole. (B) Western blotting under non-denaturing conditions of the metal ion affinity chromatography purification process. PPM: Molecular weight standard, line 1: entry medium, line 2: unbound proteins, line 3: wash with 50 mM imidaziol, line 4: elution with 250 mM imidazole.
Ejemplo 6: Ensayo de la inhibición de la proliferación de células HUVEC por el trímero formado por el scFv H2V-DTetr. Example 6: Test of the inhibition of HUVEC cell proliferation by the trimer formed by scFv H2V-DTetr.
Se sembraron células HUVEC (4to pase) en placas de 96 pocilios (103 células/pocilio) en medio RPMI-1640, 2% Suero fetal bovino, heparina (50 ug/mL). Las células se incubaron durante 16 horas a 37°C, C02 5%. Después de la incubación se realizaron 2 lavados con medio RPMI_1640 sin suero, y se le adicionó a las células medio RPMI libre de suero conteniendo hVEGF 121 purificado (10 ng/mL) y cantidades crecientes del anticuerpo trimérico H2V-DTetr purificado. Después de una incubación de 72 horas a 37 °C se eliminó el medio de cultivo y las células se tiñeron con una solución de violeta cristal 0,5% y metanol 20%. El efecto de inhibición se obtuvo mediante la lectura de las placas a 562 nm. Se tomó como 100 % de proliferación el crecimiento celular en las condiciones con hVEGF 121 sin el anticuerpo trimérico (Figura 9). HUVEC cells (4th pass) were seeded in 96-well plates (10 3 cells / well) in RPMI-1640 medium, 2% Bovine fetal serum, heparin (50 ug / mL). The cells were incubated for 16 hours at 37 ° C, 5% C02. After incubation, 2 washes were performed with RPMI_1640 medium without serum, and serum-free RPMI medium containing purified hVEGF 121 (10 ng / mL) and increasing amounts of the purified H2V-DTetr trimeric antibody was added to the cells. After a 72 hour incubation at 37 ° C the culture medium was removed and the cells stained with a solution of 0.5% crystal violet and 20% methanol. The inhibition effect was obtained by reading the plates at 562 nm. Cell growth was taken as 100% proliferation under conditions with hVEGF 121 without the trimeric antibody (Figure 9).

Claims

REIVINDICACIONES. CLAIMS.
Fragmentos de anticuerpos de cadena simple (scFv) CARACTERIZADO porque comprenden regiones variables de inmunoglobulinas humanas, separadas por un péptido de unión, codificadas por las secuencias nucleotídicas H2V (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4), H6VII (SEQID5), yDUVII (SEQID6) o secuencias homologas que reconocen epitopes de VEGF-A humano que interfieren con su actividad pro-angiogénica. Single chain antibody fragments (scFv) CHARACTERIZED because they comprise variable regions of human immunoglobulins, separated by a binding peptide, encoded by the nucleotide sequences H2V (SEQID 1), B5VIII (SEQID 2), H8V (SEQID3), E8V (SEQID4 ), H6VII (SEQID5), yDUVII (SEQID6) or homologous sequences that recognize epitopes of human VEGF-A that interfere with its pro-angiogenic activity.
Anticuerpo recombinante CARACTERIZADO por que está formado por las cadenas variables según reivindicación 1, unidas a las regiones constantes de moléculas de inmunoglobulinas humanas. Recombinant antibody CHARACTERIZED in that it is formed by the variable chains according to claim 1, linked to the constant regions of human immunoglobulin molecules.
Anticuerpos recombinantes, según la reivindicación 2, CARACTERIZADO porque los dominios constantes de inmunoglobulina humana son de tipo IgGl, lgG2, lgG3 o lgG4. Recombinant antibodies, according to claim 2, CHARACTERIZED because the human immunoglobulin constant domains are of the IgGl, lgG2, lgG3 or lgG4 type.
Anticuerpo recombinante, según la reivindicación 2, CARACTERIZADO porque la secuencia codificante para la cadena polipeptídica constituida por un fragmento scFv unido por un espaciador a los dominios constantes bisagra, CH2 y CH3 de una inmunoglobulina humana. Recombinant antibody, according to claim 2, CHARACTERIZED in that the coding sequence for the polypeptide chain constituted by an scFv fragment linked by a spacer to the hinge, CH2 and CH3 constant domains of a human immunoglobulin.
Fragmento de anticuerpo tipo Fab, CARACTERIZADO porque está formado por las secuencias de descritas en la reivindicación 1, con dominios constantes de una inmunoglobulina G humana. Fragment of Fab type antibody, CHARACTERIZED because it is formed by the sequences described in claim 1, with constant domains of a human immunoglobulin G.
Fragmento de anticuerpo CARACTERIZADO porque está compuesto por las secuencias H2V,(SEQID 1), B5VIII Antibody fragment CHARACTERIZED because it is composed of the sequences H2V, (SEQID 1), B5VIII
(SEQID 2), H8V (SEQID 2), H8V
(SEQID3), E8V (SEQID3), E8V
(SEQID4), H6VII (SEQID4), H6VII
(SEQID5), y D11VII(SEQID5), and D11VII
(SEQID6) fusionados al dominio de trimerización de la tetranectina humana. (SEQID6) fused to the trimerization domain of human tetranectin.
(SEQID7) y separados por una secuencia espaciadora (SEQID8). Molécula multlmérica CARACTERIZADA porque está compuesta al menos por dos construcciones monómericas de polipeptídos formados por el dominio de trimerización de la tetranectina humana en combinación con fragmentos de anticuerpos descritos. (SEQID7) and separated by a spacer sequence (SEQID8). Multimeric molecule CHARACTERIZED because it is composed of at least two monomeric constructions of polypeptides formed by the trimerization domain of human tetranectin in combination with described antibody fragments.
8. Moléculas multiméricas CARACTERIZADA porque está compuesta al menos por dos construcciones monoméricas de polipéptidos formados por la combinación del dominio de trimerización de la tetranectina humana y cualquier secuencia polipeptídica relacionada con anticuerpos, cualquiera sea el epítope o antígeno que reconozca. 8. Multimeric molecules CHARACTERIZED because it is composed of at least two monomeric constructions of polypeptides formed by the combination of the trimerization domain of human tetranectin and any polypeptide sequence related to antibodies, whatever the epitope or antigen it recognizes.
9. Moléculas multiméricas CARACTERIZADA porque está compuesta al menos por dos construcciones monoméricas de polipéptidos formados por la combinación del dominio de trimerización de cualquier miembro de la familia de Lectinas tipo9. Multimeric molecules CHARACTERIZED because it is composed of at least two monomeric constructions of polypeptides formed by the combination of the trimerization domain of any member of the type Lectin family.
C independientemente de su origen con cualquier secuencia polipeptídica relacionada con anticuerpos, cualquiera sea el epítope o antígeno que reconozca. C regardless of its origin with any antibody-related polypeptide sequence, whatever epitope or antigen it recognizes.
10. Uso de anticuerpos recombinantes según reivindicaciones 1-6 CARACTERIZADO porque se utiliza para la fabricación de medicamentos para el tratamiento de entidades que cursan con incremento de la angiogénesis, como entidades oculares, procesos neopiásicos, inflamatorios agudos y crónicos, y procesos autoinmunes, mediante la inmunoterapia pasiva. 10. Use of recombinant antibodies according to claims 1-6 CHARACTERIZED because it is used for the manufacture of medications for the treatment of entities that present with increased angiogenesis, such as ocular entities, neopiasic processes, acute and chronic inflammatory processes, and autoimmune processes, through passive immunotherapy.
11. Uso de anticuerpos recombinantes según reivindicaciones 1-6 CARACTERIZADO porque se utiliza para la fabricación de un medicamento para el tratamiento de tumores malignos y sus metástasis mediante inmunoterapia pasiva. 11. Use of recombinant antibodies according to claims 1-6 CHARACTERIZED because it is used for the manufacture of a medicine for the treatment of malignant tumors and their metastases through passive immunotherapy.
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