Recombinant chorionic gonadotropin, method of preparation thereof, pharmaceutical composition and uses

Abstract

The present invention relates to a recombinant single-strand chorionic gonadotropin polypeptide having an amino acid sequence encoding the beta and alpha strands of horse chorionic gonadotropin linked to one or more sequences comprising glycosylation sites, which causes excellent in vitro and in vivo activity. A DNA molecule encoding said recombinant polypeptide is also disclosed, as well as an expression vector comprising said DNA molecule, a composition comprising said recombinant protein, and a method for preparing the same.

Description

Background Technology

[0001] Equine chorionic gonadotropin (eCG) is a hormone produced in the endometrial cup of pregnant mares (Christakos and Bahl, 1979). Formerly known as Pregnant Mare Serum Gonadotropin (PMSG), this hormone is commonly used in livestock activities to induce ovulation prior to artificial insemination. It is a highly glycosylated glycoprotein composed of two α- and β-heteromer chains. The alpha subunit is common to all glycoprotein hormones (LH, FSH, TSH, CG), while the beta subunit is specific to each hormone and is responsible for specificity in receptor binding; however, in the case of GC, it binds to the same receptor as LH. In the equidae (horses, donkeys, and zebras), placental CG and pituitary LH are expressed from the same gene, so their protein sequences are identical, differing only in the carbohydrate side chains, particularly those located in the beta subunit; thus, they share a common sequence and are also called eLH / CG (Sherman et al., 1992).

[0002] eLH and eCG are encoded by the same gene described above and, in addition to having two N-glycosylations in the alpha subunit, have O-glycosylated C-terminal elongation and N-glycosylations in the beta subunit (Murphy and Martinuk, 1991); however, when expressed and secreted in different tissues, their carbohydrate side chain N and O differ significantly (Smith et al. 1993, Matsui et al. 1994, Bousfield and Butnev 2001), and as a result, they have different biological capabilities in vivo due to the longer half-life and different thermal stability of eCG compared to eLH (Galet et al. 2004).

[0003] The production of eCG is currently carried out by hemorrhaging pregnant mares that secrete the hormone between 40 and 130 days of gestation (Allen and Moor, 1972). Once purified, formulated, and controlled, this hormone is used to induce artificial estrus, particularly in female goats, cattle, and pigs. Unlike in horses, where eCG possesses only luteinizing activity, it has two excellent characteristics for use in livestock farming; in other species, this hormone possesses both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activities, while, on the other hand, its half-life in the bloodstream is increased due to its quaternary structure and multiple glycosylation sites (Stewart et al., 1976; Combarnous et al., 1981). The problem to be solved

[0004] The current production process faces challenges in maximizing the purity of the final product, as well as addressing all issues related to the production and purification of extracted hormones, particularly those concerning animal maintenance and health costs. Therefore, developing alternative production methods is crucial.

[0005] The inventors know that the difference in biological activity between eLH secreted by the pituitary gland and eCG secreted by the trophoblast is fundamentally due to differences in glycan length and sialation. Furthermore, it is clear that the lack of proper glycosylation in recombinant protein production systems has so far hindered the production of recombinant eLH / CG, which has a plasma half-life sufficient to exhibit biological activity in vivo.

[0006] The document ["Expression of a single beta-alpha chain protein of horse LH / CG in the milk of transgenic rabbits and its biological activity". Galet et al, published in Molecular and Cellular Endocrinology 174 (2000) 31-40] teaches the composition of a beta / alpha chain fusion protein without the addition of a linker. The above structure is expressed in the milk of transgenic rabbits. The obtained protein has in vitro activity similar to that of the natural protein, but is inactive in vivo, and the half-life of the produced recombinant hormone is very low.

[0007] Meanwhile, MIN Kwan-Sik et al. [published in the literature ["Biological activity of tethered equine chorionic gonadotropin (eCG) and its deglycosylated mutants," Journal of Reproduction and Development, Vol. 50, No. 3, 2004] demonstrated the fusion of beta and alpha chains in CHO-K1 cells with or without mutations that reduce glycosylation, without using any linker to fuse the alpha and beta chains. These researchers evaluated only in vitro activity (cell culture) for both LH and FSH activities, which are lower in non-glycosylated mutants. Ingestion of the active hormone is possible only in vitro.

[0008] Attempts to obtain the recombinant hormone reported in the literature ["Biological activity of recombinant equine luteinizing hormone / chorionic gonadotropin (eLH / CG) expressed in Sf9 and mimic insect cell lines", Legardinier, et al., Journal of Molecular Endocrinology (2005) 34, 47-60], which showed that beta and alpha chains were individually expressed in SF9 and mimic insect cells (SF9 with 5 added glycosyltransferases), were also unsuccessful. The two eLH / CGs produced in Sf9 and mimic cells were active in in vitro biological assays of LH and FSH and showed efficacy similar to eCG, but did not show significant in vivo biological activity in follicle-stimulating hormone (FSH) or eCG-specific assays. Although recombinant eLH / CG produced in mimic cells was more glycosylated than that produced in Sf9 cells, there was no difference in in vivo activity because the terminal sialylation of the carbohydrate chain was insufficient and blood was rapidly removed.

[0009] Another group reported on the fusion of beta and alpha chains without the use of any linker in the literature ["Expression and biological activity of short-chain recombinant equine luteinizing hormone (reLH)", Jablonka-Shariff et al. Theriogenology 67 (2007) 311-320]. Expression is performed in CHO-K1 cells. They explain LH activity in vitro (testosterone production by Leydig cells) and in vivo by the peak of testosterone production in horses. However, they do not show the presence of FSH activity. Knowing that LH produced in the pituitary gland is less glycosylated than GC produced in the trophoblast, researchers argued that the recombinant product they obtain (reLH) is more similar to eLH than eCG, and therefore CHO cells lack the appropriate glycosyltransferase activity required for eCG synthesis.

[0010] Finally, Sogayar et al. reported in patent application WO 2017 / 112987 A1 the transformation of CHO dg44 cells (a CHO K1 line known for the dhfr gene with a fusion as described by Jablonka-Shariff) and selected and amplified them to select high-production clones of recombinant glycosylated eCG. They assessed that their CHO cells express all enzymes associated with glycosylation and evaluated that this would make their eCG active in vitro and in vivo (mice and cattle). However, since the proposed process requires the selection of specific glycosylated clones that happen to have a trophoblast-like profile, the stability of said clones makes the utility of said strategy for large-scale production relative.

[0011] This is why the inventors decided to modify the structure of eLH / CG to increase the likelihood of more efficient post-translational glycosylation in a recombinant expression system without changing the affinity of eLH / CG to its receptor, and to increase its hydrodynamic volume, thereby ultimately increasing its biological activity in vivo. means of solving the problem Summary of the Invention

[0012] The present invention relates to a recombinant single-strand chorionic gonadotropin polypeptide characterized in that the polypeptide has an amino acid sequence encoding the beta chain and alpha chain of horse chorionic gonadotropin linked to one or more sequences including glycosylation sites.

[0013] In a preferred embodiment, the recombinant single-strand chorionic gonadotropin polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

[0014] DNA molecules encoding recombinant polypeptides are also related embodiments of the present application. In particular, those having sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 are so.

[0015] Expression vectors containing these DNA molecules are also the subject of this application.

[0016] In one embodiment, these polypeptides are part of a pharmaceutical composition.

[0017] These recombinant proteins and compositions are useful for the manufacture of drugs for the treatment of conditions related to reproduction or ovulation in mammals, such as superovulation, ovulation failure, ovarian insufficiency, postpartum estrus induction, or hypochondriacism in mammals such as cattle, pigs, sheep, dogs, rabbits, deer, goats, and experimental animals.

[0018] Another specific example refers to a method for producing recombinant single-strand chorionic gonadotropin by transfecting a vector, expressing it in CHO cells, and selectively purifying the immunoaffinity. Brief explanation of the drawing

[0019] Figure 1 shows an expression vector that enables obtaining variants of beta / alpha fusion proteins linked by different linkers.

[0020] Figure 2 shows the measurement of in vitro gonadotropic biological activity (FSH) and a typical dose-response curve for eCG.

[0021] Figure 3 shows the measured value of luteinizing biological activity (LH) in an in vitro dose-response curve for hCG.

[0022] Figure 4 shows the in vitro biological activity measurements for the construct expressed in HEK293T cells.

[0023] Figure 5 shows measurements of in vitro FSH biological activity for constructs expressed in HEK293T or CHOKI cells.

[0024] Figure 6 illustrates the analysis results of the supernatant of expression products from HEK293T or CHOK1 cells. A 10% natural acrylamide gel is shown. 17 µg of total protein was inoculated into each lane. The H series represents those synthesized by HEK and C cells in CHOK1. BSA 5 µg and PMSG 0.25 IU. The units corresponding to the mass of 17 µg were as follows: 2H(pCG 2) 4 IU; 5H(pCG 5) 7.2 IU; 8H(pCG 8) 4.5 IU; 10H(pCG 10) 1.4 IU; 5C(pCG 5) 0.55 IU; 8C(pCG 8) 0.22 IU.

[0025] Figure 7 shows the measurement of in vitro FSH biological activity of a clone that stably produces protein.

[0026] Figure 8 shows the analysis of purified reCG protein by immunoaffinity. The inoculated eCG patterns were 10 and 1 µg for Coomassi and Western staining, and 3.5 and 0.5 µg were loaded into constructs #5 and #8 under the same conditions.

[0027] Figure 9 shows the measurements of in vitro FSH and LH biological activity of stable clones 5G and 8F.

[0028] Figure 10 shows measurements of follicle number during treatment with a device containing progesterone in a field study of cattle. Follicle number (mean) according to follicle size and treatment day (P > 0.1) of cattle treated with eCG (native; n = 13) or reCG (n = 14) during the period before removal of the device containing P4.

[0029] Figure 11 shows the measurements of the number of follicles after removal of the progesterone-containing device in a field study in cattle. Number of follicles (mean) according to follicle size and treatment day (P > 0.1) in cattle treated with eCG (native; n = 13) or reCG (n = 14) after removal of the device containing P4.

[0030] Figure 12 shows the measured number of follicles after removing the device having a P4 level in both treatment groups. Specific details for implementing the invention details Acquisition of recombinant variant of eCG alpha-beta fusion protein

[0031] Here, the inventors describe the production of a single recombinant alpha / beta chain of eCG (reCG) by adding glycosylation sites to increase the plasma half-life. The production of a single chain allows for the introduction of sequences of multiple glycosylation sites as linkers and avoids the problem of subunit assimilation / dissociation.

[0032] To this end, the inventors modified the gene sequence by adding two sequences having multiple O-glycosylation or N-glycosylation sites. The O-glycosylation sequence is characteristic of the beta chain and is found in carboxy terminal peptides (CTPs), and the N-glycosylation sequence was designed from the common sequence of mammalian N-glycosylation, which we refer to as NTP in contrast to CTPs, because it is used in amino(N) terminal peptides.

[0033] To evaluate the functionality of different linkers, expression vectors containing various constructs were prepared as described in FIG. 1. The nucleotide and amino acid sequences of each of these variants are presented in the attached sequence list:

[0034] SEQ ID NO: 1 corresponds to the polynucleotide sequence of the construct encoding NTP-beta-CTP-NTP-alpha (Construct # 2), and SEQ ID NO: 2 corresponds to its amino acid sequence. SEQ ID NO: 3 corresponds to the polynucleotide sequence of the construct encoding NTP-beta-CTP-CTP-alpha (Construct # 5), and SEQ ID NO: 4 corresponds to its amino acid sequence. SEQ ID NO: 5 corresponds to the polynucleotide sequence of the construct encoding NTP-beta-CTP-alpha (Construct # 8), and SEQ ID NO: 6 corresponds to its amino acid sequence. SEQ ID NO: 7 corresponds to the polynucleotide sequence of the construct encoding NTP-beta-NTP-alpha (Construct # 10), and SEQ ID NO: 8 corresponds to its amino acid sequence. SEQ ID NO: 9 corresponds to the polynucleotide sequence of the construct encoding CTP-beta-CTP-CTP-alpha (Construct # 6) and SEQ ID NO: 10 corresponds to its amino acid sequence. SEQ ID NO: 11 corresponds to the polynucleotide sequence of the construct encoding beta-CTP-CTP-alpha (Construct # 4) and SEQ ID NO: 12 corresponds to its amino acid sequence. Development of a Method for Determining eCG via Competitive ELISA

[0035] The capture antibody (Ab) is a rabbit anti-eCG polyclonal antibody provided by Syntex. Specific immunoglobulins were obtained by purification using an eCG affinity column covalently bound to CL agarose. The tracer was obtained by biotinylating the sialic acid residue with alkoxyamine-PEG12-biotin.

[0036] Alkaline streptavidin phosphatase (Strp-FA) was used to quantify non-displaced Biot-eCG present in the sample or standard curve.

[0037] The test is performed on a 96-well ELISA plate, and a standard curve is constructed using the Std 1 eCG Syntex standard. Alkali phosphatase concentration is determined using the substrate 4-nitrophenyl phosphate in 100 mM Tris pH 9.2, 1 mM MgCl2. Readout at 405 nm. The dynamic range of the assay is 20–0.16 ng / well. In vitro test for measuring biological activity of gonadotropin stimulation (FSH)

[0038] HEK 293T cells were transfected with a plasmid containing the bovine FSH hormone receptor transgene and the selected antibiotic neomycin. After screening for antibiotic resistance and amplifying the resistant clones, the expression of the bFSH receptor was confirmed by 125I-FSH binding in the presence or absence of 1 IU of the standard provided by INAME as a competitor to demonstrate specific binding.

[0039] Clones expressing an FSH receptor that binds to a ligand with high affinity and induces a reaction synthesizing the second messenger cAMP were transfected with a luciferase reporter construct under a CRE promoter (pGL4 Promega). In the presence of cAMP, cells carrying this construct produce luciferase in proportion to the amount of the second messenger. The enzyme luciferase converts luciferin into metabolites and light in the presence of ATP + O2. This luminescence is proportional to the concentration of the test substance with follicle-stimulating activity when the entire system is assembled. The dynamic range of the test is 12.5 - 0.4 mU / tube.

[0040] A typical dose-response curve of in vitro biological activity is shown in Figure 2. In vitro test for the biological activity of the luteotrooic

[0041] Mouse Leydig cell lines constitutively express the LH receptor prior to the response to gonadotropin stimulation in cAMP production and progesterone secretion. The current cell line was recloned with partial loss of response to LH / hCG, and the clone with the highest reactivity to hCG was selected and transfected with the CRE-Luc reporter gene, similar to the HEK cells used in the FSH activity assay.

[0042] 25,000 cells per well are seeded and pre-incubated for 48 hours to perform the test on collagen-coated P96 plates, then standard curves of hGC or samples are added and incubation is continued for 3 hours. After the stimulation time has elapsed, the culture medium is removed and reagents are added to determine luciferase activity as in the previous test. The dynamic range of the test is 50-0.8 mIU / well.

[0043] A typical dose-response curve for hCG is shown in Figure 3. In vivo biological activity measurement test

[0044] Samples were analyzed using the European Pharmacopoeia 9th Edition (Eh. Ph.9) and the Equine Chorionic Gonadotropin Monograph for veterinary use as the analysis standard.

[0045] The presence and consequent biological activity (FSH) of natural or recombinant equine chorionic gonadotropin (eCG) should cause an increase in ovarian mass in immature female rats injected with the product. This increase is determined by comparing the average weight of a pair of ovaries obtained after treatment to the weight of an untreated pair of ovaries (reference weight). The reference weight of a pair of ovaries for the strain used in the study (Sprague Dawley(sd)) is less than 25 mg.

[0046] Methodology: 0.2 ml of the sample was injected subcutaneously into selected rats.

[0047] Injections were repeated 18, 21, 24, 42, and 48 hours after the first injection (the solution was stored in a refrigerator). Animals were sacrificed between 40 and 72 hours after the last injection. Afterward, the ovaries were removed, fat, adhesions, and fallopian tubes were separated from each animal, and finally, the weight of both ovaries of each treated rat was measured.

[0048] All animal handling, injection, slaughter, extraction, washing, and ovarian weighing were performed by personnel trained in these methodologies and animal handling.

[0049] Female Sprague Dawley (sd) rats aged 21 to 28 days (age difference of 3 days or less) and weighing approximately 50-55g (±10g range) were used in the selected group. Determination of the functionality of the work A - Evaluation in HEK293T cells

[0050] To test the gonadal activity of various different reCG constructs, the HEK293T cell line was selected as a synthesis platform for recombinant proteins. This cell line is characterized by possessing the SV40 gene and T antigens that induce plasmid proliferation, which implies that transient transfection leads to abnormal expression of the recombinant protein cloned into the appropriate plasmid. This presents a disadvantage in the glycosylation pattern of the recombinant protein synthesized by this cell line, which results in a very short half-life once present in the complex system. Since activity in the system that is generally not removed is unaffected, these proteins are used to test activity in an in vitro system.

[0051] The in vitro biological activity measured for each of constructs 2, 5, 8, and 10 expressed in HEK293T cells is shown in Fig. 4. From the results of HEK cells in vitro (Fig. 4 being representative), the inventors were able to conclude that the designed fusion proteins possess both eGC activities in vitro.

[0052] On the other hand, the in vivo biological activity of these same reCG constructs (recombinants) synthesized in HEK293T cell lines was investigated. The biological activity of the samples was estimated to be 500 lU / mL based on results obtained from in vitro tests.

[0053] The results obtained show that in all cases, the average ovarian weight was less than 25 mg, which is similar to the reference weight of the strain under study.

[0054] Therefore, it is concluded that the construct synthesized in HEK cells, although biologically active in vitro, lacks biological activity in vivo. Evaluation in B - CHOK1 cells

[0055] Based on the results obtained from HEK293T cells, the development of reCG was continued using CHOK1 cells as a platform, which produce glycoproteins with longer plasma half-lives than HEK and SF9. The selected constructs were #5 and #8 because they were expressed in higher amounts in in vitro tests.

[0056] 10x10 6 A comparison of the in vitro FSH biological activity of proteins synthesized by HEK or CHO cells is shown in Figure 5.

[0057] Meanwhile, proteins present in the supernatant were analyzed on two natural polyacrylamide gels, one of which was used for total protein detection with Coomassie G250 staining and transferred to another nitrocellulose membrane for analysis using antibodies produced in rabbits against eCG natives. The results obtained are shown in Fig. 6.

[0058] The apparent size of the recombinant protein synthesized in HEK cells is similar to the pattern of the extracted eGC. This indicates that the observed lack of in vivo activity is due to the quality of the polypeptide synthesized in HEK cells, rather than the hydrodynamic volume or amount of glycosylation.

[0059] Therefore, research was continued using the recombinant protein produced in CHOK1 cells and constructs #5 and #8, which are expressed in larger amounts.

[0060] In vivo biological activity tests showed an increase in ovarian weight within the minimum stimulation value of the standard curve. The control adjustment medium did not produce values ​​other than the control.

[0061] Given the recognition of the recombinant hormone in Western blots performed using antibodies generated against eCG natives, the supernatant of transiently transfected CHO cells was purified using an affinity column for eCG #5 construct. 13 µg of protein was obtained with a total in vitro FSH activity of 61 IU. In vivo activity was measured in three animals with ovarian weights of 203.9 mg, 119.8 mg, and 172.9 mg, and the stimulation value was similar to the maximum value of the standard curve. Generation of clones that stably produce proteins

[0062] CHO K1 cells were transfected with eCG #5 and #8 constructs, and G418 antibiotic was selected to obtain cells stably retaining the transplanted gene. Once confluence was reached, hormone expression was determined by ELISA, and the system was set to detect 0.6 ng activity in a 50 µl aliquot of the supernatant.

[0063] The selection criteria for the clones were good growth and production exceeding 1.5 lU / ml, and 4 of compost #5 and 3 of compost #8 remained as candidates. The results are shown in Figure 7.

[0064] Clones 5D, 5G, 5J, and 8F were expanded to measure in vivo biological activity, and based on the results, clones 5G and 8F were selected to further characterize the recombinant proteins by affinity chromatography of the two constructs. ReCG purification by immunoaffinity

[0065] Anti-eCG immune serum generated from sheep by Syntex was used. Specific IgG was separated according to affinity using an eCG-Sepharose column, and 3 mg of anti-eCG IgG was obtained per ml of serum. A fraction of the anti-eCG was dialyzed against coupling buffer (bicarbonate-NaCl) to obtain a total of 90 mg of IgG bound to 9 ml of Sepharose-NHS.

[0066] 1 ml / cm² of 0.22 µg filtered adjusted medium 2 Chromatographed at 1 / min, washed the bed with PBS until returned to the baseline, eluted with 3 ml of glycine pH 3, and collected the absorbance peak at 280 nm. The glycine eluent pH 3 was neutralized, filtered in PBS to the correct volume, and concentrated.

[0067] 850 µg of composition #5 and 360 µg of composition #8 were obtained. These were analyzed by PAGE without a reducing agent to show total protein bands by Coomas staining, and specific bands by Western blot using a rabbit natural hormone produced by Syntex as the first antibody. The results are shown in Fig. 8.

[0068] It should be noted that in the case of recombinant hormones, the only band detected by the antibody is identical to the band stained with Coomassie. The very high weight band is likely the dimer of the fusion protein, whereas the low weight band observed in natural proteins is due to the partial dissociation of the heteromer under operating conditions using SDS but without a reducing agent.

[0069] The in vitro gonadal and luteal stimulating activities of both the protein and standard eCG Syntex were measured. The results for mass dose-response activity and luciferase activity expressed in relative luminescence units (RLU) are shown in Figure 9. The difference in inactivity between the recombinant hormone and the natural hormone is significant, and more pronounced in the case of luteinizing hormone, where the natural hormone does not saturate activity even at high doses. Study of the biological activity of stably generated protein-producing clones

[0070] CHOK1 cells were transfected with constructs #5 and #8, and cells stably possessing the transplanted gene were selected and obtained.

[0071] Clones 5D, 5G, 5J, and 8F were expanded to select clones with good growth and production, and their biological activity was measured. The results are as follows: In vivo biological activity of stable clones of reCg Sample / Clone Evaluated ability Result (mg) check biological activity single average 5D 15 IU / mL 209.3 200.2 positivity is available 183.0 208.4 5G 15 IU / mL 225.1 204.9 positivity is available 199.6 189.9 5J 15 IU / mL 231.0 215.7 positivity is available 232.1 184.0 8F 15 IU / mL 131.7 157.7 positivity is available 163.0 178.3

[0072] Based on the confirmation of positive eCG and a high degree of ovarian growth in the treated animals, it was decided to make the first approximation of the evaluation of the above sample compared to a reference weight of 25 mg.

[0073] To this end, samples from different clones were analyzed by an analytical technique based on standard 3-point eCG curves of different concentrations using a Syntex secondary ecG standard generated in Syntex and evaluated against the standard: "International Standard NIBSC Code 62 / 001 (NIBSC)".

[0074] Due to the high response in previous tests, the sample is estimated to have a theoretical capability of 30 IU / mL, and the results obtained are displayed in the format used to process the obtained raw data: Measurement of biological activity at a single PMSG Table of Ovarian Weight (mg) by Dose

[0075] It was concluded that the analyzed clones produced recombinant proteins of positive biological activity along with a significant increase in ovarian weight.

[0076] All treated animals (8 animals for each sample analyzed) responded to the treatment without exception.

[0077] Furthermore, when compared to the responses we know and obtained in animals treated with natural hormones, the responses demonstrated by the recombinant were identical, and no unusual signals or atypical behaviors were observed in the treated animals.

[0078] The capabilities of the clone based on the obtained data are as follows: ▶ 5D: 26 lU / ml ▶ 5G: 33 lU / ml ▶ 5J: 20 lU / ml ▶ 8F: 17 lU / ml Research on the purification process :

[0079] The objective was to evaluate whether the purification breaks down molecules or if there is a loss of molecules as a result of the process.

[0080] Samples of the purified 5G product were injected into three animals. The results were as follows: ▶ 203.9 mg - 172.9 mg - 119.8 mg

[0081] Since the growth value matches the value obtained from the aforementioned unrefined clone, it was concluded that eCG is not lost or degraded during refining.

[0082] In the case of Compound 8F, subsequent attempts to obtain sufficient mass for measuring in vivo biological activity were unsuccessful. Therefore, the project continued with Compound #5 only until a production method was defined. Luteinization activity study (LH) :

[0083] The biological response of 21 to 28-day-old male Sprague Dawley(sd) rats treated with sample #5 was studied.

[0084] methodology:

[0085] Male Sprague Dawley rats (sd) aged 21 to 28 days (the age difference between them was 3 days or less) and selected groups of about 60-65 g body weight (at intervals of ±10 g) were prepared.

[0086] The sample was injected subcutaneously at a volume of 0.5 ml every 24 hours for 4 consecutive days.

[0087] After sacrificing the rats 24 hours after the last injection, the seminal vesicles were removed, washed, dried on filter paper, and weighed.

[0088] The tasks of animal manipulation, injection, slaughter, extraction, washing, and weighing of the seminal vesicles were performed by trained staff at the animal care facility.

[0089] The sample solution was prepared by calculating the mass or volume to be collected based on its titer, and then making it 5,264 lU / mL in concentration.

[0090] Results: 4 animals were treated, and the weight of the seminal vesicles after treatment was as follows: ▶ 44.0 mg - 58.0 mg - 48.0 mg - 52.0 mg

[0091] Since it is known that the standard weight of seminal vesicles (untreated) in the rat species used is less than 12 mg and the reaction begins to saturate within 50 mg, the inventors were able to conclude that the presence of luteinizing activity was confirmed.

[0092] This analysis confirms the existence of dual activity of recombinant eCG, namely: follicle-stimulating activity and luteinizing activity. Measurement of Follicle-Stimulating and Luteinizing Activity of Final 5G Samples

[0093] Luteinization and follicle-stimulating activity were analyzed according to the 9th edition of the European Pharmacopoeia.

[0094] A group of 8 or more animals was analyzed using the national INAME standard of hCG and Syntex, a secondary standard of eCG. result:

[0095] Based on the above, it was concluded that the recombinant eCG derived from Compound #5 possesses both excellent activities of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), just like natural hormones. Determining the benefits of modification by amino-terminal peptides for in vivo activity

[0096] The lack of in vivo biological activity of fusion proteins using CTP of the β-chain as a linker for both eCGs synthesized in SF9 and mimic cells (Legardinier, J Mol Endo (2005) 34) and CHO K1 (Min, Endocrine Journal (1996), 43 585-593), or eCGs produced in the mammary glands of transgenic rabbits (Galet et al, Mol Cell Endocrinol 174 (2000) 31), indicates the importance of quality as well as quantity of glycosylation. Accordingly, the inventors tested two alternative constructs for #5. One is construct #4, which is a fusion of α- and β-chains fused like construct #5 but with unmodified amino terms, and the other is construct #6, which is similar to #5 in that the amino terms are modified but in this case has a CTP peptide sequence. This modification evaluates the importance of amino-terminal modifications in the same linker sequence.

[0097] CHO K1 cells were transfected with plasmids encoding constructs #4 and #6 and selected for neomycin resistance. Resistant cells were cloned by selecting and diluting the cells (clones 4C and 6A) expressing the highest amount of protein determined by ELISA. These cells were cultured in DMEN-F12 5% SFB medium, the conditioned medium was collected, the proteins were precipitated with ammonium sulfate, suspended in PBS, and dialyzed against PBS for 36–48 hours with three buffer exchanges. The concentration of the recombinant protein was determined using ELISA and in vitro biological activity stimulated by luciferase activity. eCG product 4C 13341 lU / mg eCG product 5G 4013 lU / mg eCG product 6A 4768 lU / mg

[0098] The greater in vitro biological activity of the protein synthesized by the 4C construct is similar to the results found in the fusion of the beta-alpha chain (Min, Endocrine Journal (1996), 43 585-593). In addition, in the in vivo system, if this protein exhibits activity, it was expected to be lower than that of other recombinant proteins (reCG-5G and reGC-6C).

[0099] When these proteins were tested in in vivo assays, the protein produced using the 5G construct having an NTP peptide at the amino terminus showed an activity of 5477 lU / mg, whereas both the construct having a CTP peptide at the amino terminus and the unmodified construct (6A and 4C, respectively) did not increase the ovarian weight of the treated animals.

[0100] These data clearly demonstrate that modifying the amino terminus of the fusion protein of alpha and beta chains of horse chorionic gonadotropin with an NTP peptide increases the half-life and thus in vivo activity. Comparative field test

[0101] To evaluate the in situ behavior of the recombinant hormone, we tested whether intramuscular injection of recombinant eCG protein derived from product #5 (reCG #5) at an estimated dose of 2000 IU produced super-stimulation and super-ovulation responses, which were the same as the results obtained by injecting 2000 IU of eCG Native (Novormon 5000, Syntex).

[0102] Progesterone (P4) levels on days 3 and 7 after ovulation are similar in cattle treated with a dose of 2000 IU of eCG Native or reCG #5.

[0103] This test was conducted at the Santa Julia Zootechnics Station of the Catholic University of Cordoba (UCC) in May, June, and July 2019.

[0104] Fourteen open Angus / Heriford cattle aged 3 to 5 years were used (the test was repeated for 7 animals per group, but in 2 repetitions); all cattle were provided with a corpus luteum at the start of treatment (periodical). The body condition (BCS) was mean 2.5 (Scale 1-5, Image 1).

[0105] The animals were allowed to eat freely twice a day, with high-quality alfalfa rolls and 15 kg of chopped corn silo supplied to the feeder and delivered to a tractor equipped with a distribution mixer. Processing group

[0106] On the 10th day of treatment, a total of 18 cattle were treated with a dose of 500 µg of chloroprostenol (Cyclase, Syntex), and heat detection was performed after the application of PGF. 14 cattle that showed fever after PGF administration were selected for inclusion in the study.

[0107] All 14 cattle were treated with an intravaginal device (DIB, 1 g of P4, Syntex) and 2 mg of estradiol benzoate (Gonadiol, Syntex) on day 0 of treatment so that the appearance of a new follicular wave would be synchronized around day 4 of treatment (Bo et al, 1994).

[0108] On the 4th day, cattle were divided into two groups and administered 2000 IU of eCG Native (eCG Group, n = 7) or a product produced by Syntex (reCG Group #5, n = 7) at a dose of 350 µg, which was estimated to be equivalent to 2000 IU of eCG Native. All injection volumes were 10 ml, and the injections were administered deep intramuscularly.

[0109] On day 6.5 of treatment, a dose of 500 µg of PGF (Cyclase, Syntex) was administered, and a second dose was administered on day 7, at which time the device containing P4 (DIB 1g, Syntex Argentina) was removed. One day later (day 8), a dose of 100 µg of GnRH (Gonasyn, Syntex) was administered.

[0110] After 20 days (2nd replication - 10 days) from the end of the first iteration (17 days), cattle were processed using the same protocol, but in this case, cattle from the eCG Native group were processed into reCG #5, and vice versa. In this way, all cattle passed through both processing groups. Replication 1 date Processing date active May 10th 10th day PGF US May 20th Day 0 DIB + 2 mg EB US May 24th Day 4 2000 IU eCG o reCG #5* US May 25th Day 5 US May 26th Day 6 US May 26th Day 6.5 PGF US May 27th Day 7 x DIB + PGF US May 28th Day 8 GnRH US May 29th Day 9 US May 30th 10th day US May 31st Day 11 June 3rd Day 14 US + blood June 6th Day 17 US + blood Duplication 2 date Processing date active June 26 10th day PGF US July 6th Day 0 DIB + 2 mg EB US July 10th Day 4 2000 IU Nov o reCG #5* US July 11th Day 5 US July 12th Day 6 US July 12th Day 6.5 PGF July 13th Day 7 x DIB + PGF US July 14th Day 8 GnRH US July 15th Day 9 US July 16th 10th day US July 17th Day 11 July 20th Day 14 US + blood July 23 Day 17 PGF US + blood * Dosage per serving = 10 ml - im

[0111] Ovarian ultrasound scans were performed on the dates indicated in Tables 4 and 5 above. The ovarian structures were drawn and recorded in a spreadsheet designed for this purpose.

[0112] Animals were examined using transrectal ultrasound (Chison 500; 7.5 MHz, Doppler). All follicles with a diameter greater than 3 mm were identified and measured, and plotted in relation to their location within the ovary to evaluate each change individually. The subsequent technique used was described in the work of Knopf et al. (1989) and modified by Bo (Bo et al., 1994). The data to be evaluated were as follows: the diameter of the dominant follicle and major sub-follicles, and the number of follicles > 3 mm present in the ovary. All follicles exceeding 8 mm in diameter were identified after eCG or reCG #5 injection. After the administration of GnRH, ovulation was defined as the annihilation of follicles greater than 8 mm identified in previous observations. After ovulation, the size and volume of the corpus luteum (CL) resulting from ovulation were measured (Adams et al., 1992).

[0113] Meanwhile, as shown in Tables 4 and 5, blood samples were collected on days 14 and 17 of treatment to measure progesterone (P4) levels after ovulation.

[0114] To measure plasma progesterone levels, blood samples were collected by puncturing the jugular veins of all cattle. For each animal, a disposable needle (18G) was used, and blood was collected from a sterile 10 ml glass tube with a rubber cap. Samples were always collected in heparin tubes, centrifuged immediately after collection, and the plasma was double-frozen (A or B) at -20°C and formally identified according to the cattle tag number, processing date and time, and A or B.

[0115] The samples were treated using the Immuno chemo luminececia method (ECLIA, COBAS Module e601, Roche).

[0116] The average number of follicles according to size (8–10 mm, 10–12 mm, 12–14 mm, and > 14 mm) and treatment day (day 5, day 6, day 7, day 8, day 9, and day 10) was compared by ANOVA.

[0117] The average number of ovulations per treatment day was compared using the same statistics.

[0118] In the same way, P4 levels were compared on days 14 and 17 of the protocol. In all cases, treatment effects, replication, and their interactions were considered. Infostat software was used (Di Rienzo et al., 2018).

[0119] result:

[0120] The cattle in the eCG group of clone 2 were excluded from the study on the 6th day for treatment of respiratory disease. These cattle were treated according to the normal procedures of the research center under the supervision of the attending veterinarian.

[0121] No difference was found in the number of follicles (by size) across all treatment days (P>0.1). There was no replication effect (P>0.07) or replication interaction* group (P>0.4). Table 6. Number of follicles (mean ± SEM) according to follicle size and treatment day in cattle treated with eCG (Native; n = 13) or reCG # 5 (n = 14) Group = P>0.10 Replication = P>0.07 Group * Replication = P>0.4

[0122] These results are also shown in Figure 10, where information regarding the number of follicles (classified by size) during treatment with an intravaginal device (days 5-7 of the protocol) can be observed. It can be observed from the graph that the amount of follicles present in both groups is not different.

[0123] In the same way, the number of follicles after removing the device containing progesterone in Fig. 11 can be determined.

[0124] Total diagnosed ovulation (follicle > 8 mm loss) occurred between days 9, 10, and 11 of treatment (only one animal in the eCG group ovulated on day 11). There was no difference in the number of ovulations between the two treatment groups (P = 0.83; Table 3). Additionally, there was no replication effect (P = 0.06) or replication group interaction (P = 0.85).

[0125] No difference was found between the two treatment groups at the mean level of P4 on days 14 and 17 (P>0.7). No replication effect was found either (P>0.9). The results are shown in the following table. Table 7. P4 levels in cattle treated with 2000 IU or an equivalent dose of eCG on treatment days 14 and 17. P4 Day 14 P4 Day 17 Mean ± SEM Mean ± SEM eCG (n=13) 45.92 ± 8.67 76.45 ± 16.29 eCG-like (n=14) 49.66 ± 8.40 77.73 ± 18.54 P> 0.7

[0126] Figure 12 shows the levels and sampling dates of P4 in both groups.

[0127] Based on the results obtained, the inventors were able to conclude that reCG #5 injected into test animals produced gonadal stimulating activity similar to that produced by eCG Native (Novormon). This is because the super-stimulating response observed in cattle of both groups was similar before and after the removal of the progesterone-containing device, and the number of ovulations observed after the removal of the device was similar.

[0128] On the other hand, since the follicle growth rates were similar in both groups, the inventors could infer that the half-lives of the two products were similar; however, specific tests to determine the half-lives must be performed in the future.

[0129] The ovulation rate found using 2000 IU of eCG in this study was similar to that reported by other authors using the same dose in biologically similar cattle (Alfuraiji et al., 1993; Goulding et al., 1996).

[0130] There was no difference in P4 levels between days 14 and 17 of treatment, indicating that the function and amount of corpora lutea present in each treatment group were equivalent.

[0131] Finally, and based on similar responses obtained, the inventors believe that even when using the reCG #5 used in this study at a lower dose (400 IU), it can increase the pregnancy rate obtained in the FTAI program in anestrus beef cows and dairy cows. That is, when used for that purpose, it is possible to produce the same effect as Native eCG.

Claims

Claim 1 A recombinant single-strand chorionic gonadotropin polypeptide, wherein the polypeptide comprises amino acid sequences of a beta chain and an alpha chain of horse chorionic gonadotropin linked to one or more sequences comprising glycosylation sites, wherein the one or more sequences comprising glycosylation sites comprise an N-glycosylation sequence (NTP) linked to the N-terminal region of the beta chain, and the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO:

8. Claim 2 A DNA molecule characterized by encoding the recombinant single-stranded chorionic polypeptide described in claim 1. Claim 3 A DNA molecule according to paragraph 2, characterized by having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:

7. Claim 4 An expression vector characterized by comprising a DNA molecule described in paragraph 2 or 3. Claim 5 A pharmaceutical composition for treating a condition related to reproduction or ovulation in a mammal, characterized by comprising the recombinant chorionic gonadotropin described in claim 1 and a pharmaceutically acceptable carrier, wherein the condition related to reproduction or ovulation in the mammal is hyperovulation, ovulation failure, ovarian hypofunction, postpartum estrus induction, or hypotesticularity. Claim 6 delete Claim 7 A pharmaceutical composition according to claim 5, characterized in that the mammal is selected from cattle, pigs, sheep, dogs, rabbits, deer, goats, and experimental animals. Claim 8 A method for producing a recombinant single-strand chorionic gonadotropin, characterized by including transfecting a mammalian cell other than a human with the vector described in claim 4. Claim 9 A method of manufacture according to claim 8, characterized in that the mammalian cells other than the human are CHO cells. Claim 10 A method of manufacturing according to claim 9, further comprising the step of purifying a recombinant single-chain chorionic gonadotropin by immunoaffinity. Claim 11 In claim 1, the recombinant single-strand chorionic gonadotropin polypeptide is intended to be used to treat a condition related to reproduction or ovulation in mammals, wherein the condition related to reproduction or ovulation in mammals is hyperovulation, ovulatory failure, ovarian hypofunction, postpartum estrus induction, or hypochondriacism. Claim 12 A recombinant single-strand chorionic gonadotropin polypeptide according to claim 11, characterized in that the mammal is selected from cattle, pigs, sheep, dogs, rabbits, deer, goats, and experimental animals. Claim 13 delete

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