RU2669787C2 - Means for treatment of disease with macular oedema due to excessive vegf-a expression - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, namely, to ophthalmology, and is intended for the treatment of a disease accompanied by edema of the macula due to increased expression of VEGF-A. Polypeptide contains ranibizumab supplemented with F(ab')-fragment and contains on the heavy chain a hinje-site with cysteine residues to form disulfide bonds. Said polypeptide is introduced into a vitreous body. Further, the polypeptide may comprise polypeptide sequences selected from the CH3 domain of the antibody, unstructured polypeptides, including HAP polypeptide sequences S(G₄S)₁₆ and S(G₄S)₂₀, or a combination thereof.

EFFECT: use of the invention makes it possible to enhance the ease of use of anti-VEGF-A therapy for edema of the macula.

1 cl, 4 dwg, 11 tbl, 14 ex

Description

Technical field

The invention relates to medicine and can be used in the pharmaceutical industry for the manufacture of a medicament for the treatment of a disease accompanied by macular edema due to increased expression of vascular endothelial growth factor VEGF-A.

State of the art

The loss of vision associated with the pathogenetic mechanism of neoangiogenesis, which is based on increased expression of VEGF-A and a number of other growth factors, is becoming an increasingly urgent problem worldwide, such diseases include the wet form of age-related macular degeneration, diabetic retinopathy, myopic CVD, macular edema due to retinal vein occlusion, etc. According to the World Health Organization, age-related macular retinal degeneration (AMD) is one of the most common causes of blindness and ear vision problems in people over 59 in economically developed countries. So, in about 10% of patients aged 66 to 74 years, AMD is detected, in patients aged 75 to 85 years, the frequency of this disease increases to 30%. Age-related macular degeneration of the retina is divided into two types - exudative (wet form) and non-exudative (dry form). The dry form is about 90% of cases of AMD and is characterized by a slow decrease in visual acuity, the appearance of colloid formations (drusen) on the retina and degradation of the pigment epithelium, there are known cases of the transition of the dry form of AMD to wet [Solomon SD, Lindsley K, Vedula SS, Krzystolik MG, Hawkins BS. Anti-vascular endothelial growth factor for neovascular age-related macular degeneration. Cochrane Database Syst Rev. 2014 Aug 29; 8: CD005139]. The wet form is about 10% of AMD, characterized by the rapid development of the disease, retinal neovascularization, hemorrhages and often leads to vision loss [Comprehensive Ophthalmology. Ed. by A.K. Khurana. New Age International (P) Ltd., Publishers, New Delhi, 2007]. The incidence of AMD in the Russian Federation is 15: 1000 of the population and in 54.4% of cases is the cause of serious visual impairment, and in 22.9% of cases is the cause of blindness [Agarkova D.I. 2013. Mathematical algorithmization of the diagnosis of age-related macular degeneration. Basic research No. 5, p. 17-22 .; Rein DB, Wittenborn JS, Zhang X, Honeycutt AA, Lesesne SB, Saaddine J; Vision Health Cost-Effectiveness Study Group. Forecasting age-related macular degeneration through the year 2050: the potential impact of new treatments. Arch Ophthalmol. 2009 Apr; 127 (4): 533-40].

Diabetic retinopathy (DR) is one of the main complications of diabetes mellitus (DM) of the first and second type, the number of patients which is growing steadily every year. Thus, according to WHO experts, in 1995 there were 135 million patients with diabetes, and already in 2001 their number reached 175.4 million, by 2025 this number will increase to 300 million, and by 2030 it will reach 366 million people. The prevalence rate of DR among patients with diabetes is 45.8%. The most severe form of eye damage is proliferative diabetic retinopathy, characterized by an uncontrollable growth of pathological tissue structures in the retina, which appear in approximately 10-40% of all patients with diabetes, which, despite treatment, quickly and steadily leads to loss of vision in 2% of cases and 10% of cases - to severe visual impairment. Over the past decade, there has been an increase in the incidence of DR, which has now become the main cause of irreversible blindness, especially among people of working age, which creates serious medical and social problems in many countries of the world.

The most important pathogenetic factor in the wet form of AMD, DR and macular edema due to occlusion of the retinal veins is the vascular endothelial growth factor - VEGF-A (vascular endothelial growth factor). The VEGF family includes several ligands that differ structurally and functionally: VEGF-A, VEGF-B, VEGF-C, VEGF-D and P1GF. VEGF-A is the main factor stimulating the growth and neoplasm of blood vessels, it has a mitogenic effect and promotes the migration of endotheliocytes. VEGF-A, in turn, combines several structurally related proteins that are products of alternatively spliced mRNAs - VEGF165, VEGF121 and VEGF110. The main isoforms of VEGF-A that determine its activity are the isoforms VEGF165 and VEGF121. VEGF-B also contributes to angiogenesis, VEGF-C is involved in lymphangiogenesis during embryonic development, as well as in the functioning of differentiated adult lymphatic endotheliocytes. VEGF-D stimulates the growth of lymphatic and vascular endothelium. P1GF is involved in angiogenesis, wound healing, and the inflammatory response, [Ohr M. and Kaiser P. Aflibercept in wet age-related macular degeneration: a perspective review. Therapeutic Advances in Chronic Disease. 2012.3 (4), 153-161].

Medicines that inhibit the binding of VEGF-A to its receptors, such as ranibizumab (Lucentis®) and Eyelea®, are the main and most effective drugs used to treat wet forms of AMD, myopic CNV, diabetic retinopathy, and macular edema due to retinal vein occlusion. The intravitreal (IVT) use of such drugs not only slows down the development of these diseases, but can also slightly improve vision [BLA 125156; Summary Review 125387 Orig1s000.]. The first drug for anti-VEGF therapy in the form of intravitreal injections, certified in Russia for use in ophthalmology, was ranibizumab, which made a real revolution in the treatment of AMD and became the "gold standard". In June 2006, it was approved by the American Medicines Control Agency (FDA) as a unique treatment for age-related macular degeneration, and in 2008 it was registered in Russia.

Although the currently available drugs for IVT injection are highly effective, the technique of performing this manipulation itself is often accompanied by significant complications of the organ of vision for patients, we give an excerpt from the instructions for the use of lucentis: “very often - intraocular inflammation, vitreous inflammation, detachment vitreous humor, retinal hemorrhage, visual impairment, eye pain, vitreous destruction, conjunctival hemorrhage, eye irritation, sensory a foreign body in the eye, increased lacrimation, blepharitis, dry eyes, hyperemia of the eye, itching in the eye, increased intraocular pressure; often - retinal dystrophy, retinal dysfunction, retinal detachment, retinal tearing, retinal pigment epithelium exfoliation, retinal pigment epithelial tearing, decreased visual acuity, vitreous hemorrhage, vitreous dysfunction, uveitis, iritis, iridocyclitis, cataract, subcapsular cataract, clouding of the posterior capsule, point keratitis, corneal damage, inflammation of the anterior chamber of the eye, blurred vision, hemorrhages at the injection site, eye hemorrhage, conjunctivitis, allergic conjunctivitis, discharge from the eye, photopsy, photophobia, discomfort in the eye, swelling of the eyelids, pain in the eyelid, conjunctival hyperemia, blindness, endophthalmitis, creeping ulcer of the cornea, bleeding in the anterior chamber of the eye, keratopathy, adhesions of the iris, deposits on the r cornea, the formation of extension lines (striae) on the cornea, pain at the injection site, redness at the injection site, increased eye sensitivity, eyelid irritation. " As a result, the current trend of developers of new anti-angiogenic drugs for IWT injection is the creation of drugs with an increased half-life from the vitreous body, which would reduce the frequency of IWT injections, thereby reducing the likelihood of unwanted side effects and improving the quality of life of patients taking these drugs [Wells J. et. Al. 2016. Two-Year Results from a Comparative Effectiveness Randomized Clinical Trial. Ophthalmology, 1-9.].

Lucentis ^® (ranibizumab) is a Fab fragment of a humanized monoclonal antibody against human VEGF-A (IgG1 (κ)) and effectively blocks the interaction of the main VEGF-A isoforms - VEGF ₁₆₅ , VEGF ₁₂₁ and VEGF ₁₁₀ with VEGF receptors of the first and second types, [ Hutton-Smith LA et. al. 2016. A Mechanistic Model of the Intravitreal Pharmacokinetics of Large Molecules and the Pharmacodynamic Suppression of Ocular Vascular Endothelial Growth Factor Levels by Ranibizumab in Patients with Neovascular Age-Related Macular Degeneration. Molecular Pharmaceutics. DOI: 10.1021 / acs.molpharmaceut.Sb00849]. Eyelea ^® is a chimeric protein based on the VEGF receptor domains (3rd domain of VEGFR1 and 2nd domain of VEGFR2) and the Fc fragment of an IgG1 antibody, [Wells J. 2016. Aflibercept, Bevacizumab, or Ranibizumab for Diabetic Macular Edema. Two-Year Results from a Comparative Effectiveness Randomized Clinical Trial. American Academy of Ophthalmology]. The molecular weight of Lucentis ^® is 50 kDa, the molecular weight of Eyelea ^® is 115 kDa, including oligosaccharide chains. Lucentis ^® and Eyelea ^® are prescribed in the following mode - first three injections in a row (1 time per month), then according to indications. According to data published in the journal of the American Society of Ophthalmologists, according to the results of 2-year comparative randomized studies of the effectiveness of Eyelea ^® , Avastin ^® and Lucentis ^® , the median number of injections for the 2nd year of therapy (according to indications) was 5, 6 and 6, respectively, and for 2 years - 15, 16 and 15, respectively, with comparable drug efficacy, [Wells J. et. Al. 2016. Two-Year Results from a Comparative Effectiveness Randomized Clinical Trial. Ophthalmology, 1-9].

The elimination of preparations from the vitreous body during intravitreal administration occurs through the anterior chamber of the eye or through the choroid of the posterior chamber of the eye, while the removal of high molecular weight compounds from the vitreous occurs mainly through the anterior chamber of the eye by diffusion with a stream of aqueous humor and, to a much lesser extent degree, through the choroid of the eye due to the existence of a blood-ophthalmic barrier. The diffusion rate depends on the molecular weight of the molecule. Hutton-Smith L. et al. Have developed a mathematical mechanistic model of the pharmacokinetics of macromolecules with intravitreal administration, the basic calculations of which are in good agreement with experimental data from preclinical and clinical studies of ranibizumab and bevacizumab, in which it has been shown that an increase in the time of exposure of macromolecules in the vitreous can be achieved by increasing the molecular weight [Hutton-Smith LA et.al. 2016. A Mechanistic Model of the Intravitreal Pharmacokinetics of Large Molecules and the Pharmacodynamic Suppression of Ocular Vascular Endothelial Growth Factor Levels by Ranibizumab in Patients with Neovascular Age-Related Macular Degeneration. Molecular Pharmaceutics. DOI: 10.1021 / acs.molpharmaceut.Sb00849].

Preparations for intravitreal administration should have a low systemic effect. The systemic availability of macromolecules during intravitreal administration due to the presence of a blood-phthalmic barrier is reduced, but nevertheless a small fraction of the drug (about 1%) enters the blood [BLA 125156, Summary Review 125387Orig1s000], to reduce the systemic effect, the developers of ranibizumab used the Fab format of the molecule due to its rapid elimination from the bloodstream because Fab does not interact with FcRn. The Fc fragment of antibodies stabilizes molecules and increases their half-life from the bloodstream; therefore, this approach is widely used to create chimeric proteins [Czajkowsky, D., Shao, Z., Pleass, R. 2012. Fc-fusion proteins: new developments and future perspectives . EMBO Mol. Med., 4, 1015-1028.], Including for Eyelea ^® , [Wells J. 2016. Aflibercept, Bevacizumab, or Ranibizumab for Diabetic Macular Edema. Two-Year Results from a Comparative Effectiveness Randomized Clinical Trial. American Academy of Ophthalmology]. Other methods of protein modification are known, which increase both the molecular weight of molecules and physical dimensions (hydrodynamic volume), and their lifetime in blood plasma. [Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives, First Edition. Edited by Roland Kontermann. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA.]. One such modification is the chemical modification of proteins using polyethylene glycols (PEGs), [Veronese, F., Pasut, G. 2005. PEGylation, successful approach to drug delivery. Drag Discovery Today, v / 10, No. 21, 1451-58]. However, the adverse properties of pegylated proteins are the chemical heterogeneity of the modified molecules due to the heterogeneity of the structure of the modifying agent and the non-selective (random) mechanism of this process, as well as the accumulation of PEG derivatives in the body that are not subject to biodegradation.

There are other methods of protein modification, which are based not on chemical modification, but on the creation of genetic constructs (HA), as a result of translation of which target proteins are expressed that are fused with modifying polypeptides, giving the original proteins the desired properties. These polypeptides include:

- artificial polypeptides that contain N-glycosylation sites and repeats from the residues of glycine, aspartic acid and glutamic acid prevail [US 20090298762];

- polyanionic polypeptides from glutamic acid residues (84 and 173 residues) [US 20020169125, AU 2002252429, WO 02077036];

- C-terminal peptide (CTP) of the β-subunit of human chorionic gonadotropin containing 28 amino acid residues forming four O-glycosylation sites [US 20120015437]; One or more copies of CTP attached to the C-terminus of the molecules significantly prolongs the lifetime of therapeutic proteins in the blood plasma [US 2012035101, US 2012015437, US 2012004286, US 2011286967, US 2010317585, US 2010081614, US 5759818]. The use of CTP technology is limited by the need to obtain modified proteins exclusively in animal cells that ensure correct O-glycosylation;

- unstructured polypeptides.

A method for prolonging therapeutic protein molecules using unstructured polypeptides involves the genetic hybridization of proteins with highly soluble, chemically and immunologically inert unstructured polypeptides (NPs). Such NPs in aqueous solutions under physiological salt, temperature, and buffer conditions are resistant to folding (the formation of a tertiary structure) and maintain a stable disordered type of “ball”, the hydrodynamic volume of which depends only on the number of amino acid residues in the composition of the NP, the intrinsic size (diameter) of the modified protein and conformational flexibility of NP. In this regard, NPs can mediate a significant increase in the hydrodynamic volume of conjugated proteins, causing weakening of renal filtration and lengthening the lifetime of proteins in blood plasma, [Binder U., Skerra A. 2012. Half - Life Extension of Therapeutic Proteins via Genetic Fusion to Recombinant PEG Mimetics . Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives, First Edition. Edited by Roland Kontermann. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA].

The use of NPs for protein prolongation has advantages over chemical modification using PEG, in particular:

- proteins modified by this technology can be expressed both intracellularly and secreted in various types of cells, including bacteria and yeast, which makes it possible to significantly reduce the cost and speed up their production and purification;

- modifying NPs have extremely low immunogenicity and, like PEG, are able to screen foreign proteins from recognition by the immune system;

- modifying NPs are biodegradable, which significantly reduces the risk of toxicity or accumulation in any organs (for example, in the kidneys or liver) or cells (for example, in macrophages);

- The genetically encoded sequence of NPs guarantees the immutability and accuracy of protein modification.

In general terms, these characteristic features are characteristic of three known types of NPs that can prolong the action of proteins up to 100 times:

1) XTEN polypeptides containing a random alternation of 6 amino acid residues of alanine, glutamic acid, glycine, proline, serine and threonine [EP 2402754, US 20110312881, WO 2011123830, WO 2011123813, US 20110151433, US 20100323956, WO 201O0101502, 2748314, WO2007103515, US 7846445, US 7855279];

2) PAS polypeptides containing proline, alanine and serine residues [WO 2011144756, NZ 580670, US20100292130, EP 2173890, WO 2008155134;

3) HAP polypeptides that are glycine-rich sequences [WO 2007103515] containing glycine (G) and serine (S) residues.

The prolongation of the action of IFN due to the use of NP PAS has been shown, the smallest size of which was about 200 amino acid residues [WO 2008155134 A1]. The prolongation of the action of IFN alpha-2 person through the use of HAP polypeptides S (G ₄ S) ₁₆ and S (G ₄ S) ₂₀ [RU 2515913 C1] is shown.

However, unstructured polypeptides not only increase the life span of a therapeutic protein in the blood, but can lead to screening of active sites and a decrease in their target biological activity. In addition, the prior art shows an increase in the lifespan of a therapeutic protein modified with an unstructured polypeptide in the blood only.

Thus, there is still an urgent need to solve the problem of creating a preparation of an antibody fragment with an increased exposure time in the vitreous body and high binding affinity for the target.

Disclosure of the invention

The present invention solves the problem of creating a preparation of an antibody fragment with an increased exposure time in the vitreous body and high binding affinity for the target.

To create a drug with an increased exposure time in the vitreous, the concept of a mathematical mechanistic model of the pharmacokinetics of drugs with intravitreal administration was taken as the basis - an increase in the weight of the therapeutic protein. However, it was decided to move away from the traditional approach of increasing the mass of therapeutic protein by modifying it with NP, PEG. Using ranibizumab as an example, the authors of the present invention investigated the possibility of increasing the weight of a therapeutic fragment of an antibody by adding an F (ab ') fragment. The result was a genetic engineering construct of F (ab ') ₂ -ranibizumab for eukaryotic expression containing a hinje site with cysteine residues on the heavy chain to form disulfide bonds. We used the F (ab ') ₂ -ranibizumab molecule as a basic structure for the development of other variants of molecules modified with various polypeptides in order to increase their mass and hydrodynamic volume. As the polypeptides that modify the F (ab ') ₂ -ranibizumab, we used either the IgG1 immunoglobulin Fc fragment or the IgG1 immunoglobulin Fc fragment CH3 domain or unstructured hydrophilic polypeptides (NGPs) fused either to the hinje region or to CH3 -domain of the Fc fragment of immunoglobulin IgG1, or with the light chain of the ranibizumab molecule.

Numerous studies of the obtained product showed that F (ab ') ₂ -ranibizumab has an increased exposure time in the vitreous body and at the same time is characterized by an unexpectedly high affinity for VEGF-A compared to ranizumab.

Thus, an object of the present invention is a polypeptide containing a F (ab ') ₂ fragment of a therapeutic antibody for administration to the vitreous body for the treatment and / or prophylaxis of a disease in respect of which said antibody has therapeutic activity.

Said protein is preferably ranibizumab, and said disease is any disease accompanied by macular edema due to increased expression of VEGF-A.

Moreover, it will be apparent to a person skilled in the art that for a subsequent increase in the mass and duration of action of the F (ab ') ₂ fragment of a therapeutic antibody of the invention, it can be supplemented with other polypeptide sequences. For example, such polypeptide sequences can be the CH3 domain of an antibody and / or NP, including the HAP polypeptide sequences S (G ₄ S) ₁₆ and S (G ₄ S) ₂₀ and others according to the NP mentioned in the "Background" section. Therefore, the F (ab ') ₂ fragment of a therapeutic antibody of the invention may further include polypeptide sequences selected from, inter alia, S (G ₄ S) ₁₆ , S (G ₄ S) ₂₀ , the CH3 domain, or a combination thereof.

The use of the invention improves the convenience of anti-VEGF-A therapy for macular edema due to the increased exposure time in the vitreous body and high affinity of binding of the anti-VEGF-A polypeptide to the target, which will significantly reduce the number of administrations of the therapeutic agent.

Brief Description of the Drawings

FIG. 1. The binding of proteins based on the F (ab ') ₂ fragment of ranibizumab with recombinant human endothelial growth factor rhVEGF ₁₂₁ , determined by ELISA.

FIG. 2a. The dependence of the median concentration of the protein being determined on the day of vitreous sampling.

FIG. 2b. The dependence of the median concentration of the protein being determined on the day of sampling of aqueous humor.

FIG. 3. The dependence of the median concentration of the detected protein on the time of sampling of blood.

The implementation of the invention

The stages of obtaining and characterizing the inventive hybrid proteins based on F (ab ') ₂ -ranibizumab.

Stage 1. Obtaining genetic engineering constructs for eukaryotic expression. Using routine genetic engineering methods, synthetic DNA sequences encoding the heavy and light chains of the F (ab ') ₂ fragment of ranibizumab (Lucentis®) were cloned into plasmid vectors designed for expression of proteins in eukaryotic cells.

Using routine genetic engineering methods, the DNA sequences encoding the heavy chain of the F (ab ') ₂ heavy chain of ranibizumab (Lucentis®) and the third heavy chain constant domain (CH3 domain) of a human immunoglobulin IgG class I subclass obtained by PCR using PCR were correctly merged corresponding original primers.

The construction of plasmid DNA designed for expression in eukaryotic cells of the proteins Lucentis-HC-NHP, Lucentis-LC-NHP and Lucentis-HC-CH3-NHP fused to an unstructured hydrophilic peptide (NGP) was carried out using routine genetic engineering methods using auxiliary plasmids containing sequences encoding the corresponding proteins, and the correct fusion of the sequences of these proteins with sequences encoding NGP. Target expression plasmids were obtained by transferring fragments of accessory plasmids encoding the proteins Lucentis-HC-NHP, Lucentis-LC-NHP and Lucentis-HC-CH3-NHP to plasmid vectors designed for expression of proteins in eukaryotic cells.

Stage 2. Obtaining clones producing proteins based on eukaryotic cells. The prepared HICs were used for electroporation of eukaryotic cells and the creation of producer clones based on them.

Stage 3. Cultivation of producer clones and protein isolation.

The cultivation of clones producing target proteins based on the F (ab ') ₂ fragment of ranibizumab was carried out in a disposable wave bioreactor in the fed-batch mode (periodic cultivation with recharge).

Protein isolation was carried out in several stages - the affinity chromatography stage using Capto L sorbents (GE Healthcare) or KappaSelect (GE Healthcare), the hydroxyapatite ion exchange chromatography stage (CHT-I, Bio-Rad) and SP-sepharose (GE Healthcare).

Stage 4. The study of physico-chemical and functional properties in vitro and in vivo.

The isolated proteins were characterized on the basis of the F (ab ') ₂ fragment of ranibizumab by means of PAGE-electrophoresis with sodium dodecyl sulfate, HPLC gel filtration, ELISA, plasmon surface resonance and in the test of inhibition of VEGF-A-induced proliferation of HUVEC cells (primary culture of endothelocytes human umbilical vein).

Step 5. Evaluation of the pharmacokinetic parameters of proteins based on the F (ab ') ₂ fragment of ranibizumab with intravitreal administration to rabbits.

The aim of the study was a comparative assessment of the pharmacokinetics and toxicokinetics of proteins based on the F (ab ') ₂ fragment of ranibizumab and Lucentis ^® (Novartis) after a single intravitreal administration to Soviet chinchilla rabbits.

Further, the implementation of the invention is shown by specific examples, which should not be construed as limiting in relation to the created invention. The specialist in the prior art it is clear that without the cost of inventive creativity can be developed additions and changes relative to the disclosed in the following examples within the framework of the present invention.

Example 1. Construction of a recombinant plasmid pRA1654 encoding the heavy chain F (ab) 2 fragment of ranibizumab (Lucentis®).

A DNA fragment is synthesized having open “sticky” ends identical to the “sticky” ends created by HindIII restriction endonucleases at the 5'-end and XbaI at the 3'-end, and the coding polypeptide

corresponding to the heavy chain of the F (ab) 2-fragment of ranibizumab (Lucentis®) with the N-terminal leader sequence (italics) of the coagulation factor FVII removed from the composition of the target protein during transport of clone producer cells from the cytoplasm.

Recombinant plasmid pRA1654 is constructed on the basis of a plasmid vector for expression of proteins in eukaryotic cells, which incorporates the following structural elements:

structural elements of the CMV promoter (CMV enhancer, EF-1α promoter, EF-1α intron A); bovine growth hormone polyadenylation signal (bGH poly (A) signal); high copy plasmid replication origin (ori ColE1 / pMB1 / pBR322 / pUC); a gene encoding β-lactamase providing resistance to ampicillin, carbenicillin and similar antibiotics (AmpR, AmpR promoter); origin of bacteriophage replication f1 (f1 ori); early promoter of the SV40 virus (SV40 promoter); SV40 virus replication origin (SV40 ori); the PuroR gene encoding puromycin N-acetyltransferase providing resistance to puromycin (alternatively may contain HygR encoding hygromycin B phosphotransferase providing resistance to hygromycin B, or NeoR / KanR encoding aminoglycoside phosphotransferase from Tn5, which provides resistance to canomycin and Geneticin®); a synthetic polyadenylation signal (poly (A) signal); as well as unique HindIII and XbaI restriction endonuclease recognition sites located in series.

To this end, the synthetic DNA fragment is cloned into a selected vector, split at the HindIII and XbaI sites so that the cloning sites are restored as part of the resulting plasmid. The result is a recombinant plasmid pRA1654 containing a cloned synthetic DNA fragment encoding the heavy chain F (ab) 2-fragment of ranibizumab (Lucentis®)

Example 2. Construction of a recombinant plasmid pRA1655 encoding the light chain F (ab) 2 fragment of ranibizumab (Lucentis®).

A DNA fragment is synthesized having open “sticky” ends identical to the “sticky” ends created by HindIII restriction endonucleases at the 5'-end and XbaI at the 3'-end, and the coding polypeptide

corresponding to the light chain of the F (ab) 2-fragment of ranibizumab (Lucentis®) with the N-terminal leader sequence (italics) of the coagulation factor FVII removed from the composition of the target protein during transport of clone producer cells from the cytoplasm.

Recombinant plasmid pRA1655 is constructed on the basis of a plasmid vector for expression of proteins in eukaryotic cells, which incorporates the structural elements mentioned in Example 1.

To this end, the synthetic DNA fragment is cloned into a selected vector, split at the HindIII and XbaI sites so that the cloning sites are restored as part of the resulting plasmid. The result is a recombinant plasmid pRA1655 containing a cloned synthetic DNA fragment encoding the light chain of the F (ab) 2 fragment of ranibizumab (Lucentis®).

Example 3. Construction of the recombinant plasmid pMT1605 encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1.

To obtain the recombinant plasmid pMT1605 encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1, 2 polymerase chain reactions were carried out.

Reaction 1. As a matrix, the recombinant plasmid pRA1654 was used (the preparation is described in Example 1), synthetic oligonucleotides were used as seed crystals:

St3_for

and

OMT907

;

;

the resulting fragment with the SmaI restriction endonuclease recognition site inserted at the 3'-end encodes the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®).

Reaction 2. As a matrix, we used a laboratory recombinant plasmid encoding the third constant domain of the human immunoglobulin heavy chain of subclass IgG class 1 (CH3 domain of human IgG1), synthetic oligonucleotides were used as seeds:

oMT908

and

oRA405

;

;

the obtained fragment with the DraI restriction endonuclease recognition site inserted at the 5'-end encodes the human IgG1 CH3 domain.

The recombinant plasmid pMT1605 was constructed on the basis of a plasmid vector for the expression of proteins in eukaryotic cells, which incorporates the structural elements mentioned in Example 1.

The fragment obtained during Reaction 1 was cleaved using restriction endonucleases HindIII and SmaI; and the fragment obtained during Reaction 2, using DraI and XbaI. The obtained fragments are purified using spin columns and used in a three-component (vector + insert 1 + insert 2) ligation reaction with a selected vector that is cleaved at the HindIII and XbaI sites so that the HindIII and XbaI cloning sites are restored as part of the resulting plasmid. The result is a recombinant plasmid pMT1605 containing a cloned DNA fragment encoding the heavy chain F (ab) 2 fragment of ranibizumab (Lucentis®) fused to the CH3 domain of human IgG1 encoding a polypeptide

the corresponding heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to the CH3 domain of human IgG1 with an N-terminal leader sequence (italics) of the coagulation factor FVII removed from the target protein during transport of clone producer cells from the cytoplasm.

Example 4. Construction of the recombinant plasmid pMT1616 encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1 fused to NGP.

To obtain the recombinant plasmid pMT1616 encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1 fused to NGP, an auxiliary plasmid No. 1 was obtained.

Recombinant auxiliary plasmid No. 1 is constructed on the basis of any laboratory plasmid vector containing unique recognition sites for the restriction endonucleases HindIII and XbaI, as well as structural elements such as the origin of replication of high copy plasmids (ori ColE1 / pMB1 / pBR322 / pUC); a gene encoding β-lactamase providing resistance to ampicillin, carbenicillin and similar antibiotics (AmpR, AmpR promoter); origin of bacteriophage replication f1 (f1 ori); and not containing the recognition site of the restriction endonuclease SapI, for example, a modified plasmid pBK685.

To this end, the recombinant plasmid pMT1605 encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to the CH3 domain of human IgG1 is cleaved using HindIII and XbaI restriction endonucleases and the resulting restriction fragments are separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-1. Fragment-1 is cloned into a selected vector, split at the HindIII and XbaI sites so that the cloning sites are restored as part of the resulting plasmid. The result is a recombinant accessory plasmid No. 1A or pMT1608 containing a cloned DNA fragment encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1.

Reaction 3. To introduce the recognition site of the restriction endonuclease SapI in the 3'-end region for 4 bp PCR was performed before the stop codon. The recombinant plasmid pMT1608 was used as a matrix; synthetic oligonucleotides were used as seeds:

oMT909

and

OMT910

;

;

the obtained fragment with the SapI restriction endonuclease recognition site inserted at the 3'-end encodes a C-terminal modified heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1.

The DNA fragment obtained during Reaction 3 was digested using restriction endonucleases HindIII and XbaI; purified using spin columns and used in the ligation reaction with the selected vector (pBK685), split at the HindIII and XbaI sites so that, as part of the resulting plasmid (auxiliary plasmid No. 16), the HindIII and XbaI cloning sites were restored.

To obtain a recombinant auxiliary plasmid No. 1, auxiliary plasmid No. 16 was cleaved at the SapI restriction endonuclease recognition site. At the same time, the recombinant plasmid encoding NGP was digested at the SapI restriction endonuclease recognition site and the resulting restriction fragments were separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-2. Fragment-2 was cloned into an auxiliary plasmid No. 16 digested at the SapI site. The resulting recombinant auxiliary plasmid No. 1 contains a cloned DNA fragment encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1 fused to NGP.

The recombinant plasmid pMT1616 is constructed on the basis of a plasmid vector for expression of proteins in eukaryotic cells, which incorporates the structural elements mentioned in Example 1.

To this end, recombinant accessory plasmid No. 1 was digested at the recognition sites with HindIII and XbaI restriction endonucleases, and the resulting restriction fragments were separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-3. Fragment-3 was cloned into a selected vector for eukaryotic expression, cleaved at the HindIII and XbaI sites so that the cloning sites were restored in the resulting plasmid. The result is a recombinant plasmid pMT1616 containing a cloned DNA fragment encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) with the CH3 domain of human IgG1 fused to NGP.

Recombinant plasmid pMT1616 encodes a polypeptide

the corresponding heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to the CH3 domain of human IgG1, fused to the NHP (bold), with the N-terminal leader sequence (italics) of the coagulation factor FVII removed from the composition of the target protein in the process transport from the cytoplasm of producer clone cells.

Example 5. Construction of the recombinant plasmid pMT1612 encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to NGP.

To obtain the recombinant plasmid pMT1612 encoding the heavy chain F (ab) of the 2-fragment of ranibizumab (Lucentis®) fused to the non-hypertensive region, an auxiliary plasmid No. 2 was obtained.

Recombinant auxiliary plasmid No. 2 receive as well as the recombinant auxiliary plasmid No. 1 described in Example 4.

To this end, the recombinant plasmid pRA1654 from Example 1 encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) was digested with restriction endonucleases HindIII and XbaI and the resulting restriction fragments were separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-4. Fragment-4 was cloned in the vector pBK685 described in Example 4, split at the HindIII and XbaI sites so that the cloning sites were restored in the resulting plasmid. The result is a recombinant accessory plasmid No. 2A (pMT1606) containing a cloned DNA fragment encoding the heavy chain F (ab) 2 fragment of ranibizumab (Lucentis®).

Reaction 4. To introduce the recognition site of the restriction endonuclease SapI in the 3'-end region for 4 bp PCR was performed before the stop codon. The recombinant plasmid pMT1606 was used as a matrix; synthetic oligonucleotides were used as seeds:

OMT909

and

oMT911

,

,

the obtained fragment with the SapI restriction endonuclease recognition site inserted at the 3'-end encodes a C-terminated heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®).

The DNA fragment obtained during Reaction 4 was digested using restriction endonucleases HindIII and XbaI; purified using spin columns and used in the ligation reaction with the selected pBK685 vector cleaved at the HindIII and XbaI sites so that the HindIII and XbaI cloning sites are restored as part of the resulting plasmid (auxiliary plasmid No. 2b).

To obtain a recombinant auxiliary plasmid No. 2, auxiliary plasmid No. 2b was digested at the SapI restriction endonuclease recognition site. At the same time, the recombinant plasmid encoding NGP was digested at the SapI restriction endonuclease recognition site and the resulting restriction fragments were separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-5. Fragment-5 is cloned into an auxiliary plasmid No. 2b, split at the SapI site. The resulting recombinant accessory plasmid No. 2 contains a cloned DNA fragment encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to NGP.

The recombinant plasmid pMT1612 is constructed on the basis of a plasmid vector for expression of proteins in eukaryotic cells, which incorporates the structural elements mentioned in Example 1.

To this end, recombinant accessory plasmid No. 2 was digested at the recognition sites with HindIII and XbaI restriction endonucleases, and the resulting restriction fragments were separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-6. Fragment-6 is cloned into a selected vector for eukaryotic expression, cleaved at the HindIII and XbaI sites so that the cloning sites are restored in the resulting plasmid. The result is a recombinant plasmid pMT1612 containing a cloned DNA fragment encoding the heavy chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to NGP.

Recombinant plasmid pMT1612 encodes a polypeptide

the corresponding heavy chain of the F (ab) 2-fragment of ranibizumab (Lucentis®), fused to the NHP (bold), with the N-terminal leader sequence (italics) of the coagulation factor FVII removed from the composition of the target protein during the transport of clone cells from the cytoplasm producer.

Example 6. Construction of recombinant plasmids pMT1613, pMT1614 and pMT1615 encoding the light chain F (ab) 2-fragment of ranibizumab (Lucentis®) fused to NGP.

To obtain the recombinant plasmid pMT1613, pMT1614, and pMT1615 encoding the light chain F (ab) of the 2-fragment of ranibizumab (Lucentis®) fused to NGP, auxiliary plasmid No. 3 was obtained.

Recombinant auxiliary plasmid No. 3 receive as well as recombinant auxiliary plasmids No. 1 and No. 2 described in Examples 4 and 5.

To this end, the recombinant plasmid pRA1655 from Example 2 encoding the light chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) was digested with restriction endonucleases HindIII and XbaI and the resulting restriction fragments were gel separated. A smaller DNA fragment is eluted from the gel and is called fragment-7. Fragment-7 was cloned in the vector pBK685 described in Example 4, split at the HindIII and XbaI sites so that the cloning sites were restored in the resulting plasmid. The result is a recombinant accessory plasmid No. 3A (pMT1607) containing a cloned DNA fragment encoding the light chain F (ab) 2 fragment of ranibizumab (Lucentis®).

Reaction 5. For the introduction of the recognition site of the restriction endonuclease SapI in the 3'-end region for 4 bp PCR was performed before the stop codon. The recombinant plasmid pMT1607 was used as a matrix; synthetic oligonucleotides were used as seeds:

OMT909

and

oMT912

,

,

the obtained fragment with the SapI restriction endonuclease recognition site inserted at the 3'-end encodes the C (end) modified light chain of the F (ab) 2 fragment of ranibizumab (Lucentis®).

The DNA fragment obtained during Reaction 5 was digested using restriction endonucleases HindIII and XbaI; purified using spin columns and used in the ligation reaction with the selected vector pBK685 cleaved at the HindIII and XbaI sites so that the cloning sites of HindIII and XbaI are restored as part of the resulting plasmid (auxiliary plasmid No. 3b).

To obtain a recombinant auxiliary plasmid No. 3, auxiliary plasmid No. 3b was digested at the SapI restriction endonuclease recognition site. At the same time, the recombinant plasmid encoding NGP was digested at the SapI restriction endonuclease recognition site and the resulting restriction fragments were separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-8. Fragment-8 is cloned into an auxiliary plasmid No. 3b, split at the SapI site. The resulting recombinant accessory plasmid No. 3 contains a cloned DNA fragment encoding the light chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to NGP.

The recombinant plasmid pMT1613 is constructed on the basis of a plasmid vector for the expression of proteins in eukaryotic cells, which incorporates the structural elements mentioned in Example 1.

To this end, recombinant accessory plasmid No. 3 was digested at the recognition sites with HindIII and XbaI restriction endonucleases, and the resulting restriction fragments were separated in a gel. A smaller DNA fragment is eluted from the gel and is called fragment-9. Fragment-9 is cloned into a selected vector for eukaryotic expression, split at the HindIII and XbaI sites so that the cloning sites are restored in the resulting plasmid. The result is a recombinant plasmid pMT1613 containing a cloned DNA fragment encoding the light chain F (ab) 2-fragment of ranibizumab (Lucentis®), fused with NGP.

The recombinant plasmid pMT1614 is constructed on the basis of a plasmid vector for the expression of proteins in eukaryotic cells, which differs from the vector containing the structural elements mentioned in Example 1 in that the PuroR element encoding puromycin N-acetyltransferase, which provides resistance to puromycin, is replaced HygR encoding hygromycin B phosphotransferase providing resistance to hygromycin B.

To obtain the target expression vector pMT1614, fragment-9 is cloned into a selected eukaryotic expression vector, cleaved at the HindIII and XbaI sites so that the cloning sites are restored in the resulting plasmid. The result is a recombinant plasmid pMT1614 containing a cloned DNA fragment encoding the light chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to NGP.

The recombinant plasmid pMT1615 is constructed on the basis of a plasmid vector for the expression of proteins in eukaryotic cells, which differs from the vector containing the structural elements mentioned in Example 1 in that the PuroR element encoding puromycin N-acetyltransferase, which provides resistance to puromycin, is replaced on NeoR / KanR, encoding the aminoglycoside phosphotransferase from Tn5, which provides resistance to neomycin, kanamycin and G418 (Geneticin®).

To obtain the target expression vector pMT1615, fragment-9 is cloned into the selected eukaryotic expression vector, split at the HindIII and XbaI sites so that the cloning sites are restored in the resulting plasmid. The result is a recombinant plasmid pMT1615 containing a cloned DNA fragment encoding the light chain of the F (ab) 2 fragment of ranibizumab (Lucentis®) fused to NGP.

Recombinant plasmids pMT1613, pMT1614 and pMT1615 encode the polypeptide

the corresponding light chain of the F (ab) 2-fragment of ranibizumab (Lucentis®), fused to the NHP (bold), with the N-terminal leader sequence (italics) of the coagulation factor FVII, removed from the composition of the target protein during transport of clone cells from the cytoplasm producer.

Example 7. Obtaining clones producing proteins based on eukaryotic cells.

The prepared HICs were used to create producing clones based on eukaryotic cells. For this, the preparations of the plasmids described in examples 1-6 encoding the light and heavy chains of the corresponding protein were mixed, and eukaryotic cells were transfected by electroporation. The transfected cells were selected by cultivation on a selective antibiotic. After obtaining stable pools of producer cells of the corresponding proteins, they were cloned, the culture fluid of single clones was tested by solid-phase ELISA on immobilized recombinant human VEGF-A, and clones with the maximum productivity of the target proteins based on the F (ab ') ₂ fragment of ranibizumab were selected.

Example 8. Cultivation of producer clones and protein isolation.

The cultivation of clones producing target proteins based on the F (ab ') ₂ fragment of ranibizumab was carried out in a disposable wave bioreactor in the fed-batch mode (periodic cultivation with recharge). SFM4CHO (HyClone, USA) was used as the basic nutrient medium, and BalanCD CHO Feed 2 (Irvine Scientific, USA) was used as a feed. The cultivation duration was 10 days. At the end of the cultivation, cell suspensions were carried out through a 3-stage clarification filtration and the target proteins were extracted from the culture fluid.

At the first stage of purification of all protein variants based on the F (ab ') ₂ fragment of ranibizumab, affinity chromatography was used using Capto L sorbents (GE Healthcare) or KappaSelect (GE Healthcare). Both sorbents specifically bind the light chain of immunoglobulins of the IgG-kappa type for its variable (Capto L) or constant (KappaSelect) part. Sorbents were balanced with phosphate-buffered saline, clarified culture fluid was applied, washed with a balancing buffer from unbound impurities, then target proteins were eluted with a buffer containing 50 mM histidine, pH 2.5. For all proteins, a similar isolation efficiency was shown when using both sorbents.

In the second stage of purification, a multimodal sorbent hydroxyapatite (CHT-I, Bio-Rad) was used, which is often used to purify antibodies from related impurities and residual proteins of producer cells. The sorbent was pre-equilibrated with a solution of 50 mM histidine, pH 7.0, then the protein eluates were applied after the affinity step, diluted with water to a conductivity of 3-5 mS / cm, the sorbent was washed with a balancing buffer, then with a buffer containing 50 mM histidine, 1 M sodium chloride, pH 7.0. Target proteins were eluted with a linear gradient of sodium phosphate (pH 7.0) to a concentration of 300 mM. All fractions obtained at this chromatographic stage were analyzed by HPLC gel filtration and PAGE using sodium dodecyl sulfate to select fractions containing the desired forms of proteins. Hydroxyapatite purification made it possible to efficiently remove low molecular weight related impurities, as well as residual impurity proteins.

If necessary, additional purification of the target protein was carried out by filtration through a SP-sepharose cation exchange sorbent (GE Healthcare) at pH 5.5 and conductivity of 4-6 mS / cm, as well as concentration and diafiltration of samples into the required buffer on cut-off Amicon Ultra concentrators (Millipore) by molecular weight 30-50 kDa. At the end of the isolation, the purity of the protein was at least 90% of the target form of the protein according to electrophoresis and HPLC gel filtration.

Example 9. Physico-chemical properties of proteins determined by HPLC gel filtration.

A total of 7 different proteins were obtained based on the F (ab ') ₂ fragment of ranibizumab, and the isolated proteins were characterized by HPLC gel filtration. For this, an Alliance 2695 HPLC system (Waters) was used, with a UV / Visible Detector 2487 (Waters) and a Superdex200 column (Increase 10/300 GL, Lot. 10226065, No. 0128; GE Healthcare). The phosphate-saline isotonic buffer was used as the mobile phase. Table 1 shows the symbol of the proteins, their nominal and apparent molecular weight obtained by HPLC gel filtration.

Variants of proteins based on the F (ab ') ₂ fragment of ranibizumab T3, T4, T5, T6 and T7 have a large apparent molecular weight according to HPLC gel filtration, exceeding their nominal molecular weight from 2.61 to 4.96 times, which due to the presence of NGP in their structure.

Example 10. The interaction of proteins with recombinant VEGF-A (rhVEGF121) person, determined by ELISA.

Protein preparations based on the F (ab ') ₂ fragment of ranibizumab were characterized by ELISA by binding to the recombinant human vascular endothelial growth factor rhVEGF ₁₂₁ manufactured by MBC Generium LLC. For this, rhVEGF ₁₂₁ manufactured by MBC Generium LLC was added to the wells of ELISA plates of 100 μl at a concentration of 1 μg / ml in carbonate-bicarbonate buffer (pH 9.5) and sorbed overnight at + 4C. Residual free plastic binding sites were blocked with PBS-Tha solution (PanEco, Cat. No. E4100-110) for 1 hour at room temperature, after which 100 μl of protein solutions diluted with PBS-0.05% Tw20 were added to the wells serial 3-fold dilutions from 100 to 0.002 nM. The plates were incubated for 1 hour, unbound antibodies were washed off, protein complexes with rhVEGF ₁₂₁ were detected using biotinylated polyclonal rat antibodies to ranibizumab manufactured by MBC Generium LLC and streptavidin horseradish peroxidase conjugated (Abcam, ab7403). TMB (USA, Party: TMBPD10222-2-10273-A) was used as a substrate for horseradish peroxidase, the reaction was stopped with a solution of 2M H ₂ SO ₄ . The development of the reaction was evaluated by the optical density measured at a wavelength of 450 nm. The results of the study are presented in Figure 1.

Table 2 shows the averaged data on the affinity of GNR049 candidates (EC50, nM) obtained using ELISA.

All proteins based on the F (ab ') ₂ fragment of ranibizumab interact with the recombinant human endothelial growth factor rhVEGF ₁₂₁ , while variants of proteins that have NGP give lower optical density at saturating concentrations compared to proteins without NGP, and the apparent protein affinity carriers of NGP, decreases with increasing proportion of NGP polypeptide in the molecular weight.

Example 11. The interaction of proteins with recombinant VEGF-A (rhVEGF121) person, determined by surface plasmon resonance.

The kinetic characteristics of the interaction of proteins based on the F (ab ') ₂ fragment of ranibizumab were evaluated by surface plasmon resonance on a Biacore T200 instrument. In the first series of experiments, the kinetic characteristics of the interaction of the commercial drug Lucentis® (Novartis) and the F (ab ') ₂ fragment of ranibizumab obtained at LLC MBC Generium were compared. For this, the recombinant human protein rhVEGF ₁₂₁ manufactured by MBC Generium LLC and the recombinant commercial rabbit VEGF-A (Kingfisher Biotech, Inc., Cat. No. RPO923U-100) were covalently immobilized on a CM-5 chip, then Lucentis® solutions (Novartis) were passed ) and F (ab ') ₂ fragment of ranibizumab in HBS-EP + buffer. Working concentrations 0.74, 2.22, 6.67, 20, 60 nM. Association 210 sec in HBS-EP + buffer. Dissociation 14400 sec (4 hours) in 20 mM Na-Acetate buffer, 150 mM NaCl, 3 mM EDTA, 0.005% Tween20, pH 4.5. Final regeneration with 50 mM Gly-HCl solution, pH 2.5. Due to the use of low pH in dissociation, apparent KDs were obtained for variants of Lucentis with human VEGF121. Apparent K _D was calculated from the mathematical model of protein-protein interaction of 1: 1. The data are shown in table 3.

A change in the format of the molecule from the Fab fragment to the F (ab ') ₂ fragment with a constant primary structure led to an increase in apparent affinity by about 10 times, such a significant increase in the affinity of the F (ab') ₂ fragment of ranibizumab was unexpected.

In the second series of experiments, the kinetic characteristics of the interaction of other protein variants based on the F (ab ') ₂ fragment of ranibizumab with the recombinant human vascular endothelial growth factor rhVEGF ₁₂₁ by the surface plasmon resonance method on a Biacore T200 instrument according to the above procedure were evaluated. The data are shown in table 4.

As can be seen from the data presented in table 4, the kinetic characteristics of the interaction of proteins based on the F (ab ') ₂ fragment of ranibizumab with the recombinant human vascular growth factor rhVEGF ₁₂₁ are the association rate constants of the complex K _a (K _on ) and the dissociation rate of the complex K _d (K _off ), as well as the equilibrium dissociation constant of the complex K _D , are not inferior to all variants of dimeric molecules, and even surpass those characteristics for ranibizumab. At the same time, RUmax for proteins that have an NGP in their structure is significantly lower than for proteins without NGP, while this indicator should increase with increasing molecular weight, which we observe for the ranibizumab monomer and dimer — for the monomer RUmax is 52 , 83, and for dimers - 78.07 and 96.27. A significant decrease in RUmax for proteins with NGP can be explained by both a small fraction of active molecules in the protein preparation and the fact that the modified NGP molecules have a larger hydrodynamic volume, and when complexed with rhVEGF ₁₂₁ covalently immobilized on the chip substrate, the NGP shields the neighboring immobilized rhVEGF molecule ₁₂₁ , creating the effect of decreasing the density of immobilized rhVEGF ₁₂₁ molecules available for interaction, which leads to a decrease in RUmax. The explanation of the RUmax reduction phenomenon for protein preparations with NGP in the analysis on Biacore T200 required us to conduct an experiment with the formation of a complex of each of the proteins with rhVEGF ₁₂₁ in solution, since there are no steric difficulties in complex formation in the solution.

Example 12. The formation of a complex of proteins based on the F (ab ') ₂ fragment of ranibizumab with recombinant human vascular endothelial growth factor rhVEGF ₁₂₁ in solution.

When analyzing the kinetic characteristics of the interaction of drugs with rhVEGF ₁₂₁ on Biacore T200, a significant decrease in RUmax was observed for modified NGP proteins based on the F (ab ') ₂ fragment of ranibizumab, which could indicate steric difficulties in the interaction of proteins carrying NGP with the antigen immobilized on the substrate due to their large hydrodynamic radius, and the fact that some proportion of the protein in the preparations is not active. For this, an additional analysis of the activity of the prepared preparations was carried out, studying their interaction with rhVEGF ₁₂₁ in solution at an equimolar ratio. Proteins were mixed with rhVEGF ₁₂₁ in an equimolar ratio in PBS solution, incubated overnight at + 4 ° C, then HPLC gel filtration was performed on a Superdex S200 column, complexation efficiency was evaluated by the peak area of free rhVEGF ₁₂₁ compared to the corresponding parameter for the preparation Lucentis ^® (Novartis). The data are shown in table 5.

As can be seen from the data presented in table 5, the peak area of free rhVEGF ₁₂₁ at an equimolar ratio of Lucentis ^® (Novartis) and rhVEGF ₁₂₁ is 34% of the peak area of rhVEGF ₁₂₁ without adding any binding agent. With the exception of T5, the peak area of free rhVEGF ₁₂₁ for which was 57%, the corresponding indicator for other proteins based on the F (ab ') ₂ fragment of ranibizumab varies from 16% to 37%, which proves the high antigen-binding ability of the developed molecules, not inferior to that characteristic for lucentis.

Example 13. Inhibition of rhVEGF121-induced proliferation of HUVEC cells with proteins based on the E (ab ') ₂ fragment of ranibizumab.

A comparative determination of the biological activity of protein preparations based on the F (ab ') ₂ fragment of ranibizumab and the Lucentis ^® preparation (Novartis) was performed in the rhVEGF ₁₂₁ -induced proliferation inhibition test of the primary culture of human umbilical vein endothelial cells (HUVEC, Lonza, Cat. No. CC2517) . For this, HUVEC I cells of passage (P1) (Lonza, Cat. No. CC2517) were thawed and plated in complete growth medium EGM-2 (Endothelial Growth Medium) in a T25 vial precoated with 0.1% gelatin solution. After the cells reached a monolayer (P2), they were removed from the plastic by treatment with a nektase solution and plated in a T75 vial, also previously coated with a solution of 0.1% gelatin. When the cells reached maximum density (P3), they were plated in gelatin-coated wells of a 96-well flat-bottom plate in the amount of 10 thousand cells per well in complete growth medium EGM-2 (passage 3). The next day, HUVEC cells were washed gently twice with sterile PBS and incubated in EBM supplemented with 0.2% FBS (test medium) for 2.5-4 hours. Protein preparations based on the F (ab ') ₂ fragment of ranibizumab, Lucentis ^®, and rhVEGF ₁₂₁ were diluted in test medium in accordance with the experimental design. Then, each of the tested preparations in a series of 8 consecutive 3-fold dilutions from 100 to 0.05 nM was incubated with rhVEGF ₁₂₁ (125 ng / ml) for 2 hours, after which pre-incubated solutions were added to HUVEC cell plates according to the experimental design. The following controls were additionally made on each plate — wells containing only rhVEGF ₁₂₁ (125 ng / ml); wells containing complete growth medium and wells with test medium. All combinations of test substances were added to the cells in triplicates. The plates were placed in a C02 incubator and incubated at 37 ° C and 5% CO ₂ for 72 hours. After incubation, to assess cell viability, XTT reagent (Roche, Cat. No. 11465015001) was added to the wells of the plates according to the manufacturer's instructions and the plates were further incubated at 37 ° C and 5% CO ₂ for 4-6 hours. The optical density was measured at a wavelength of 492 nM and a reference wavelength of 690 nM. The percentage inhibition of VEGF-A-induced proliferation of HUVEC cells was calculated by the formula:

where "average Abs. VEGF121 - average Abs. VEGF-0" corresponds to the difference between the average optical density in the wells with the addition of rhVEGF ₁₂₁ (125 ng / ml) and the average optical density in the wells with the test medium, i.e. maximum proliferation of HUVEC cells during the test;

"Abs. VEGF121 / Tx" corresponds to the optical density in the well containing the test protein at a certain concentration and rhVEGF ₁₂₁ , and

"average Abs. VEGF-0" corresponds to the average optical density of wells containing only test medium without the addition of growth additives.

The data obtained were calculated in GraphPad Prism version 6.0 using the 4-parameter logistic function "dose-response - inhibition", the data are presented in table 6.

As can be seen from the data presented in table 6, protein preparations based on the F (ab ') ₂ fragment of ranibizumab are not inferior in biological activity to the drug Lucentis ^® (Novartis) in the inhibition test of inhibition of rhVEGF ₁₂₁ -induced HUVEC cell proliferation in vitro. This test has a high predictive ability of the antiangiogenic efficacy of drugs in vivo, including when administered to humans. The half-maximum concentration of inhibition (IC50) of rhVEGF _121- induced proliferation of the drug Lucentis ^® (Novartis) ranged from 2.5 to 3.2 nM, the average value was 2.9 nM, and the standard deviation was 0.3 nM. The IC50 of protein preparations based on the F (ab ') ₂ fragment of ranibizumab ranged from 1.3 nM to 2.5 nM, which suggests that all proteins have high antiangiogenic activity, comparable to that for the "gold standard" in treatment ophthalmic diseases, the pathogenesis of which is due to the high activity of the vascular endothelial growth factor - ranibizumab.

Example 14. In vivo preclinical studies - a study of the pharmacokinetics and toxicokinetics of proteins based on the F (ab ') ₂ fragment of ranibizumab with a single intravitreal administration to Soviet chinchilla rabbits.

The aim of the study was a comparative determination of the main parameters of pharmacokinetics and toxicokinetics with an assessment of ophthalmotoxicity and possible total toxic effect of proteins based on the F (ab ') ₂ fragment of ranibizumab and Lucentis ^® (Novartis) with a single intravitreal administration to Soviet chinchilla rabbits. The study was carried out in NPO House of Pharmacy CJSC (Russia, Leningrad Region, Vsevolozhsk Municipal District, Kuzmolovsky City Settlement) in accordance with the basic principles of good laboratory practice and ethical standards for the protection of animals used for scientific purposes.

The objectives of the study included the study of the influence of the tested objects on the biometric parameters of the studied animals — mortality and dynamics of body weight; study of the influence of the tested objects on the clinical condition of the eye structures of the studied animals; ophthalmoscopic assessment of the influence of test objects on the fundus of experimental animals. Tables 7 and 8 show the main groups of animals, the dose of drugs for intravitreal administration and the main procedures performed during the study.

Materials and methods. The study was conducted on sexually mature male rabbits of the Soviet Chinchilla breed. Test objects were administered intravitreal to animals at a dose of 50 μl in each eye of the animal. The volume of administration of test objects for the study of pharmacokinetics with an assessment of ophthalmotoxicity was chosen based on bioethical principles, taking into account the invasiveness and pain of the intravitreal administration procedure, as well as on the basis of data on the maximum recommended volumes of intravitreal administration to rabbits. An intravitreal route of administration has been used since this route of administration is the intended mode of use in clinical practice.

Intravitreal injections were carried out after anesthesia of animals by intravenous administration of a mixture of Zoetil®100 preparations (Virbac, France) at a dose of 1.5 mg / kg and Rometar® (Bioveta, Czech Republic) at a dose of 1.5 mg / kg (SOP EZh-20) . A mixture of anesthetics in this dose made it possible to achieve the optimal duration of anesthesia for injections in both eyes of the animal and ensured the complete absence of a corneal reflex throughout the duration of the manipulations. For local anesthesia, a solution of local anesthetic (Inocain®, eye drops, Promed Export PVT. LTD) was added to the conjunctival sac. The sequence of manipulations when performing intravitreal injection was as follows: 1) adding a solution of mydriatic (Irifrin®, eye drops, Promed Export PVT. LTD) into the conjunctival sac, waiting 5 minutes; 2) washing the surface of the cornea with sterile saline; 3) the introduction of a solution of local anesthetic (Inocain®) in the conjunctival sac, waiting 3 minutes; 4) washing the surface of the cornea with sterile saline; 5) the introduction of Betadine, 0.5% solution (Pharmaceuticals PLC (Hungary)); 6) holding the needle of a disposable insulin syringe through the sclera at a distance of 2-3 mm from the limb towards the equator in the upper-outer quadrant; holding the needle deep into the vitreous body 2-3 mm parallel to the lens.

Inspection of animals in cages, in order to detect mortality or signs of deviation in health, was carried out daily.

Feed deprivation - animals were deprived of morning feeding before the administration and weighing of animals. Access to water was not restricted.

Body weight was recorded in the morning immediately before administration, then weekly. The procedure was carried out on the scales of a medical benchtop MT Karapuz 20 VZHA- (5g, P) bA, head. No. 488688. The smallest weighing limit is 100 g. The largest weighing limit is 20 kg. The price of calibration is 5 g. Accuracy class 3.

A clinical examination of the visible structures of the eye was carried out immediately before the start of the study, then daily. A semi-quantitative assessment of ophthalmotoxicity parameters was carried out in points. In the presence of a pathological sign, 1 point was assigned, in the absence of a sign, the score was 0. The following ophthalmotoxicity indicators were evaluated:

- Xerophthalmia (dryness of the cornea and conjunctiva of the eye, resulting from a violation of lacrimation);

- Keratitis (inflammation of the cornea of the eye, accompanied by its clouding);

- lacrimation;

- Suppuration;

- Puffiness;

- redness;

- Exophthalmos (displacement anterior to the eyeballs);

- Mydriasis (dilated pupil);

- Myosis (a significant narrowing of the pupil, a physiological reflex reaction that occurs when the eyes are illuminated with light of high brightness).

An ophthalmoscopic assessment of the condition of the fundus was carried out before the start of the study, on the 2nd, 13th and 29th days after injection of the preparations by direct ophthalmoscopy and examination of the anterior part of the eye with a magnifier (40 diopters) after the administration of Atropine (drops ophthalmic 1% (Moscow Endocrine Plant (Russia)). Pathological changes in the macula, central region of the retina, optic disc and retinal vessels were evaluated.

Blood sampling (1 ml each) was carried out in sterile tubes with EDTA at 4, 8, 24, 48, 72, 96, 192 and 384 hours after drug administration. Blood samples were centrifuged to obtain plasma. Each plasma sample was divided into two parts, placed in labeled cryostable tubes, the labeling of each tube included the number of the experimental group, number of the animal, type of bio-sample, date and time of sampling. All samples were frozen, stored at -70 ° C and transported to the Research Sponsor for further analyzes in dry ice.

Euthanasia of animals in accordance with Directive 2010/63 / EU of the European Parliament and the Council of the European Union for the Protection of Animals Used for Scientific Purposes of September 22, 2010, was carried out by overdose with anesthetics. Euthanasia was carried out using the preparations Zoletil® and Rometar®, after the animals were introduced into deep anesthesia, the drug Lidocaine was administered intravenously (Dalchimpharm (Russia)). This type of euthanasia is humane and does not contradict regulatory documents, is accompanied by a minimum of pain, suffering and distress, and was carried out by competent employees.

After animal euthanasia, the entire vitreous and aqueous humor were collected. Each biological sample was divided into two parts, placed in labeled cryostable tubes, the labeling of each tube included the number of the experimental group, the number of the animal, the type of biosample, the date and time of sampling. All samples were frozen, stored at -70 ° C and transported to the Research Sponsor for further analyzes in dry ice.

Statistical analysis of animal body mass data - the data was checked for compliance with the law of normal distribution using the Shapiro-Wilk test. In the case of a normal distribution, the mean value and the standard error of the mean were calculated, which, together with the n value (number of observations), are presented in the final tables. To evaluate data with signs of a normal distribution, one-way analysis of variance (ANOVA) was used, which was used to compare several samples (groups) and calculate the overall significance level of differences. Statistical analysis was performed using Statistica 6.0 software. (StatSoft, USA).

Conclusions and conclusions of NPO Dom Pharmaciya CJSC on the assessment of the ophthalmotoxicity and general toxic effect of protein preparations based on the F (ab ') ₂ fragment of ranibizumab and the comparative preparation Lucentis ^® (Novartis) with a single intravitreal administration to Soviet chinchilla rabbits:

1. The tested objects T1, T2, T3, T4, T5, T6, T7, T8, as well as the reference drug Lucentis®, did not affect the dynamics of the body weight of animals.

2. In all groups (T1, T2, T3, T4, T5, T6, T7, T8,) there were no statistically significant deviations from the control groups in terms of the incidence of the studied clinically significant deviations.

Evaluation results of the pharmacokinetics and toxicokinetics of protein preparations based on the F (ab ') ₂ fragment of ranibizumab with a single intravitreal administration to Soviet chinchilla rabbits obtained at MBC Generium LLC

In order to determine the concentration of drugs in biological samples of rabbits, a specific and highly sensitive method was developed in the sandwich ELISA variant based on polyclonal rat antibodies to ranibizumab obtained at MBC Generium LLC.

Briefly, polyclonal rat antibodies to ranibizumab were added to the wells of ELISA plates of 100 μl at a concentration of 1 μg / ml in carbonate-bicarbonate buffer (pH 9.5) and immobilized overnight at + 4C. Residual free plastic binding sites were blocked with PBS-Tha solution (PanEco, Cat. No. E4100-110) for 1 hour at room temperature. After that, biological samples diluted with a solution of PBS-0.05% Tw20 in serial 2-fold dilutions were added to the wells of the plates. The plates were incubated for 1 hour, unbound antibodies were washed and a solution of biotinylated polyclonal rabbit anti-ranibizumab antibodies was introduced (MBC Generium LLC). After an incubation of 1 hour, unbound antibodies were washed off and horseradish peroxidase conjugated streptavidin was added at a dilution recommended by the manufacturer (Abcam, ab7403). Incubated 40 minutes at room temperature and the horseradish peroxidase substrate was added. TMB was used as a substrate for HRP (USA, Party: TMBPD10222-2-10273-A), the reaction was stopped with a solution of 2M H ₂ SO ₄ . The reaction was evaluated by optical density measured on a Bio-Rad xMark microplate spectrophotometer at a wavelength of 450 nm. The data obtained were calculated using the Bio-Rad Microplate Manager 6 software for a microplate spectrophotometer.

The concentration of the studied preparations in the vitreous body after a single IVT administration and a preliminary assessment of the ratio of the half-lives of the vitreous preparations of proteins based on the F (ab ') ₂ fragment of ranibizumab and Lucentis ^® (Novartis)

The design of the comparative screening study included only three time points for the selection of biological samples, in accordance with the basic principles of ethical standards for the treatment of laboratory animals and minimizing the number of experimental animals, since the main objective of the study was a comparative study of the pharmacokinetic parameters in IWT with the introduction of a large panel of the studied drugs and Lucentis ^® . We did not seek to accurately determine the parameters of pharmacokinetics, such as the speed and half-life of vitreous preparations, this task would require much more laboratory animals, but only wanted to establish the ratio of these parameters for each of the studied drugs and Lucentis ^® . Vitreous samples were taken from experimental animals after euthanasia and enucleation of the eyes on the 1st day (after 4 hours), on the 14th and 30th day after IVT injection. The concentration of protein preparations based on the F (ab ') ₂ fragment of ranibizumab was determined by the method described above with respect to the corresponding calibration curve for each of the studied proteins.

Figures 2-4 show graphs of the dependence of the medians of the concentration of the determined proteins in the vitreous, aqueous humor, and blood plasma in molar terms, on the time of taking biological samples after a single IVT injection.

We calculated one of the main criteria for pharmacokinetics, reflecting the exposure of each of the studied drugs in the vitreous body, the area under each curve (AUC _IVT , nM / (ml * h), (Fig. 33), in accordance with the linear rule of trapeziums using Excel PK Solver 2.0 The following formula was used to calculate the elimination rate (clearance, Cl);

where V _{ss IVT} is the equilibrium volume of distribution in the vitreous, D _IVT is the IVT dose, T _{1/2 IVT} is the half-life from the vitreous.

Based on the assumption that the equilibrium distribution volume in the vitreous is the same for all the molecules studied and for Lucentis ^® , the following equality is obtained:

The calculation data are shown in table 9.

As can be seen from the data in table 9, the maximum exposure time in the vitreous was determined for the drug Lucentis ^® with its larger relative to other drugs, molar D _IVT - 6667 nM, molar IWT doses of other drugs were significantly (from 4.5 to 2.4 times) less than D Lucentis _IVT and, accordingly, their AUC _IVT values were less than 2.3 to 3 times lower than the AUC _IVT for Lucentis ^® . With the smallest D _IVT of T7 (less than 4.5 times the dose of Lucentis ^® ), its AUC _IVT was less than the corresponding value of Lucentis ^® only 2.5 times. The elimination rate of the studied molecules was significantly lower for protein preparations based on the F (ab ') ₂ fragment of ranibizumab modified with NGP than for the Lucentis ^® preparation and unmodified NGP protein variants. The lowest elimination rate was shown for T7. The ratio of the half-lives of the studied drugs and the Lucentis ^® preparation from the vitreous body was more than 1 for all modified NHP variants, and for variants T6 and T7 this ratio was 1.5 and 1.8, respectively. This means that the half-life of the latter is longer than the T _{1 / 2IVT of the} drug Lucentis ^{® by} at least 1.5 and 1.8 times, since the calculation of the AUC _IVT , based on which the excretion rate was calculated, was performed only up to 30 days, when the concentration of Lucentis ^{® was} no longer determined, but T6 and T7 were also reliably determined. To accurately determine the half-life of the T6 and T7 preparations from the vitreous, it is necessary to conduct an additional study with a large number of temporary selection points in the range from 1 to 45-60 days of the study.

The data presented in table 10 indicate that the maximum medians of the concentration of the drugs in aqueous humor were determined 4 hours after the IVT injection, the maximum value of the median concentration after 4 hours was observed for the drug lucentis, the minimum for the drug T7. On day 14, the concentration of drugs in all groups decreased; on day 30, only modified NGP protein variants were determined in samples of aqueous humor, which indicates that on day 30 of the study, these drugs were still contained in the vitreous, unlike lucentis and unmodified NGP proteins, since proteins enter the vitreous humor. This fact is consistent with the determination of the concentration of the studied drugs in the vitreous body. AUC _water was maximal for the drug lucentis in accordance with its D _IVT , therefore, for a more correct comparison of this indicator between the experimental groups, we calculated the AUC _water normalized relative to the corresponding D _{IVT .} -A _water , which reflects the effectiveness of the drug from the vitreous into the anterior chamber of the eye. This indicator (A _water.vl. ) Was the maximum for lucentis and the minimum for T7. The percentage of indicator A _vod.vl. from the corresponding normalized indicator in the vitreous - A _IVT was also maximum for Lucentis and minimum for T7, this indicator also reflects the effectiveness of removing drugs from the vitreous through the aqueous humor of the anterior chamber of the eye.

Table 11 presents the main pharmacokinetic parameters of the studied preparations T6 and T7, for which an increased exposure time in the vitreous body is shown, in comparison with the drug Lucentis ^® .

The calculated AUC _(plasma) for Lucentis ^® is 2.4 times greater than the corresponding value for GNR049 / T7, but the IVT dose of Lucentis ^® is 4 times higher than the D _IVT of GNR049 / T7. The values of AUC _(plasma) -A _(plasma) normalized with respect to D _IVT more informatively reflect both the ability of the drugs to enter the bloodstream and the time of their exposure in the bloodstream. The value of A _(plasma) for the T7 preparation is only 1.9 times higher than the corresponding value for the Lucentis ^® preparation, which, with the proven safety of the use of the Lucentis ^® preparation in clinical practice, most likely does not lead to undesirable effects due to the systemic effect of the T7 preparation, which represents a modified variant of the drug Lucentis ^® .

Thus, the new T7 protein is expressed in a eukaryotic system with productivity acceptable for production, has a high affinity for human and rabbit VEGF-A comparable to ranibizumab, high efficiency in inhibiting VEGF-A-induced proliferation of HUVEC cells, a previously estimated T7 half-life from the vitreous body during IVT administration to rabbits is approximately two times higher than the corresponding indicator for ranibizumab with comparable indicators of the systemic effect of these drugs.

Claims (2)

1. A polypeptide containing ranibizumab with added F (ab ') fragment and containing on the heavy chain a hinje region with cysteine residues for the formation of disulfide bonds, for the treatment and / or prevention of a disease in respect of which the antibody has therapeutic activity, when introduction into the vitreous body. 2. The polypeptide according to claim 1, characterized in that it further comprises polypeptide sequences selected from the CH3 domain of an antibody, unstructured polypeptides, including HAP polypeptide sequences S (G ₄ S) ₁₆ and S (G ₄ S) ₂₀ , or their combinations.

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