HUMAN-DERIVED RECOMBINANT FSH FOR CONTROLLED OVARIAN

Abstract

Preparations including FSH, for example recombinant FSH, for use in the treatment of infertility.

Claims

1. A composition comprising recombinant follicle stimulating hormone (FSH), wherein the recombinant FSH includes α2,3- and α2,6-sialylation and mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24% of the sialylated glycan structures are tetrasialylated glycan structures, and wherein 20% or more of the glycans comprise N-Acetylgalactosamine GalNAc.

2. The composition according to claim 1, wherein the composition further comprises a GnRH antagonist.

3. The composition according to claim 1, wherein the composition further comprises human chorionic gonadotropin (hCG).

4. The composition according to claim 1, wherein the composition further comprises a salt comprising a pharmaceutically acceptable alkali metal cation selected from Na.sup.+-salts and K.sup.+-salts, and combinations thereof.

5. A method of treating infertility in a female patient having a serum anti-müllerian hormone (AMH) level of 0.05 pmol/L or above, comprising administering to the patient a product comprising recombinant follicle stimulating hormone (FSH) at a daily dose of 15 μg to 24 μg recombinant FSH.

6. The method according to claim 5, wherein the product is administered at a daily dose of recombinant FSH selected from 15 μg, 18 μg, and 24 μg.

7. The method according to claim 5, further comprising, prior to the administering, determining the serum AMH level of the patient.

8. The method according to claim 5, wherein the recombinant FSH includes α2,3- and α2,6-sialylation.

9. The method according to claim 5, wherein the product further comprises hCG.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0079] The present invention will now be described in more detail with reference to the attached drawings in which:

[0080] FIG. 1 shows a plasmid map of the pFSHalpha/beta expression vector;

[0081] FIG. 2 shows the α2,3-sialyltransferase (ST3GAL4) expression vector;

[0082] FIG. 3 shows the α2,6-sialyltransferase (ST6GAL1) expression vector;

[0083] FIG. 4 shows % abundance sialic acid distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after engineering with α2,3-sialyltransferase;

[0084] FIG. 5 shows % abundance of glycan charge distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after engineering with α2,3-sialyltransferase;

[0085] FIG. 6 shows a comparison of concentration of inhibin-B following administration of 225 IU Gonal f (bottom line, dotted line) and 225 IU of the Example (top line, full line) of Invention;

[0086] FIG. 7 shows the effect of body weight on oocytes retrieved in the low AMH treatment group (Example 10, 10A); and

[0087] FIG. 8 shows the effect of Body weight on oocytes retrieved in the high AMH treatment group

SEQUENCE SELECTION

Human FSH

[0088] The coding region of the gene for the FSH alpha polypeptide was used to according to Fiddes and Goodman. (1981). The sequence is banked as AH007338 and at the time of construction there were no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO:1.

[0089] The coding region of the gene for FSH beta polypeptide was used according to Keene et al (1989). The sequence is banked as NM_000510 and at the time of construction there were no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO: 2

Sialyltransferase

[0090] α2,3-Sialyltransferase—The coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (α2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred herein as SEQ ID NO: 3.

[0091] α2,6-Sialyltransferase—The coding region of the gene for beta-galactosamide alpha-2,6-sialyltransferase 1 (α2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (1990). The sequence is banked as NM_003032 and referred herein as SEQ ID NO: 4.

EXAMPLES

Example 1 Construction of the FSH Expression Vector

[0092] The coding sequence of FSH alpha polypeptide (AH007338, SEQ ID NO: 1) and FSH beta polypeptide (NM_003032, SEQ ID NO: 2) were amplified by PCR using the primer combinations FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec respectively.

TABLE-US-00001 FSHa-fw (SEQ ID NO: 9) 5′-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3′ FSHa-rev (SEQ ID NO: 10) 5′-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3′ FSHb-fw (SEQ ID NO: 11) 5′-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3′ FSHb-rev (SEQ ID NO: 12) 5′-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-3′

[0093] The resulting amplified FSH beta DNA was digested with the restriction enzymes AscI and HpaI and inserted into the AscI and HpaI sites on the CMV driven mammalian expression vector carrying a neomycin selection marker. Similarly the FSH alpha DNA was digested with BamHI and NheI and inserted into the sites BamHI and NheI on the expression vector already containing the FSH beta polypeptide DNA.

[0094] The vector DNA was used to transform the DH5α strain of E. coli. Colonies were picked for amplification. Colonies containing the vector containing both FSH alpha and beta were selected for sequencing and all contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid pFSH A+B #17 was selected for transfection (FIG. 1).

Example 2 Construction of the ST3 Expression Vector

[0095] The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4 (ST3, L23767, SEQ ID NO: 3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.

TABLE-US-00002 2,3STfw (SEQ ID NO: 13) 5′-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3′ 2,3STrev (SEQ ID NO: 14) 5′-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3′

[0096] The resulting amplified ST3 DNA was digested with the restriction enzymes BamHI and AflII and inserted into the BamHI and AflII sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST3 #1 (FIG. 2) contained the correct sequence according SEQ ID NO: 3 and was selected for transfection.

Example 3 Construction of the ST6 Expression Vector

[0097] The coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6, NM_003032, SEQ ID NO: 4) was amplified by PCR using the primer combination 2,6STfw and 2,6STrev.

TABLE-US-00003 2,6STfw (SEQ ID NO: 15) 5′-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3′ 2,6STrev (SEQ ID NO: 16) 5′-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3′

[0098] The resulting amplified ST6 DNA was digested with the restriction enzymes BamHI and AflII and inserted into the BamHI and AflII sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST6 #11 (FIG. 3) contained the correct sequence according SEQ ID NO: 4 and was selected for transfection.

Example 4 Stable Expression of pFSH □+□□in PER.C6® Cells. Transfection Isolation and Screening of Clones

[0099] PER.C6® clones producing FSH were generated by expressing both polypeptide chains of FSH from a single plasmid (see Example 1).

[0100] To obtain stable clones a liposome based transfection agent with the pFSH □+□□construct. Stable clones were selected in VPRO supplemented with 10% FCS and containing G418. Three weeks after transfection G418 resistant clones grew out. Clones were selected for isolation. The isolated clones were cultured in selection medium until 70-80% confluent. Supernatants were assayed for FSH protein content using an FSH selective ELISA and pharmacological activity at the FSH receptor in cloned cell line, using a cAMP accumulation assay. Clones expressing functional protein were progressed for culture expansion to 24 well, 6 well and T80 flasks.

[0101] Studies to determine productivity and quality of the material from seven clones were initiated in T80 flasks to generate sufficient material. Cells were cultured in supplemented media as previously described for 7 days and the supernatant harvested. Productivity was determined using the FSH selective ELISA. The isoelectric profile of the material was determined by Isoelectric focusing (IEF), by methods known in the art. Clones with sufficient productivity and quality were selected for sialyltransferase engineering.

Example 5 Level of Sialylation is Increased in Cells that Over Express α2,3-Sialyltransferase. Stable Expression of pST3 in FSH Expressing PER.C6® Cells; Transfection Isolation and Screening of Clones

[0102] PER.C6® clones producing highly sialylated FSH were generated by expressing α2,3 sialyltransferase from separate plasmids (Example 2) in PER.C6® cells already expressing both polypeptide chains of FSH (from Example 4). Clones produced from PER.C6® cells as set out in Example 4 were selected for their characteristics including productivity, good growth profile, production of functional protein, and produced FSH which included some sialylation. Stable clones were generated as previously described in Example 4. Clones were isolated, expanded and assayed. The α2,3-sialyltransferase clones were adapted to serum free media and suspension conditions.

[0103] As before, clones were assayed using a FSH selective ELISA, functional response in an FSH receptor cell line, IEF, metabolic clearance rate and Steelman Pohley analysis. Results were compared to a commercially available recombinant FSH (Gonal-f, Serono) and the parental FSH PER.C6® cell lines. FSH produced by most of the clones has significantly improved sialylation (i.e. on average more FSH isoforms with high numbers of sialic acids) compared to FSH expressed without α2,3-sialyltransferase. In conclusion expression of FSH together with sialyltransferase in PER.C6® cells resulted in increased levels of sialylated FSH compared to cells expressing FSH only.

Example 6 Production and Purification Overview

[0104] A procedure was developed to produce FSH in PER.C6® cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several FSH-producing PER.C6® cell lines.

[0105] FSH from α2,3-clone (Example 5) was prepared using a using a modification of the method described by Lowry et al. (1976).

[0106] For the production of PER.C6®-FSH, the cell lines were adapted to a serum-free medium, i.e., Excell 525 (JRH Biosciences). The cells were first cultured to form a 70%-90% confluent monolayer in a T80 culture flask. On passage the cells were re-suspended in the serum free medium, Excell 525+4 mM L-Glutamine, to a cell density of 0.3×10.sup.6 cells/ml. A 25 ml cell suspension was put in a 250 ml shaker flask and shaken at 100 rpm at 37° C. at 5% CO.sub.2. After reaching a cell density of >I×I0.sup.6 cells/ml, the cells were sub-cultured to a cell density of 0.2 or 0.3×10.sup.6 cells/ml and further cultured in shaker flasks at 37° C., 5% CO.sub.2 and 100 rpm.

[0107] For the production of FSH, the cells were transferred to a serum-free production medium, i.e., VPRO (JRH Biosciences), which supports the growth of PER.C6® cells to very high cell densities (usually >10.sup.7 cells/ml in a batch culture). The cells were first cultured to >1×I0.sup.6 cells/ml in Excell 525, then spun down for 5 min at 1000 rpm and subsequently suspended in VPRO medium+6 mM L-glutamine to a density of 1×10.sup.6 cells/ml. The cells were then cultured in a shaker flask for 7-10 days at 37° C., 5% CO.sub.2 and 100 rpm. During this period, the cells grew to a density of >10.sup.7 cells/ml. The culture medium was harvested after the cell viability started to decline. The cells were spun down for 5 min at 1000 rpm and the supernatant was used for the quantification and purification of FSH. The concentration of FSH was determined using ELISA (DRG EIA 1288).

[0108] Thereafter, purification of FSH was carried out using a modification of the method described by Lowry et al. (1976). Purification using charge selective chromatography was carried out to enrich the highly sialylated forms by methods well known in the art.

[0109] During all chromatographic procedures, enrichment of the sialylated forms of FSH as claimed herein was confirmed by RIA (DRG EIA 1288) and/or IEF.

Example 7 Quantification of Relative Amounts of α2,3 and α2,6 Sialic Acid

[0110] The relative percentage amounts of α2,3 and α2,6 sialic acid on purified rFSH (Example 6) were measured using known techniques.

[0111] N-Glycans were released from the samples using PNGase F under denaturative conditions and then labelled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of charge distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column.

[0112] The relative percentages of the charged glycans were calculated from structures present in the undigested and digested glycan pools and are shown in FIG. 4 (for 8 samples). These were found to be in the ranges 50%-70% (e.g. about 60% or 65%) for α2,3 sialylation and 28 to 50%, generally 30 to 35% (e.g. about 31% or 35%), for α2,6 sialylation.

Example 8 Quantification of Relative Amounts Mono, Di, Tri and Tetra Sialylated Glycan Structures

[0113] The relative percentage amounts of mono, di, tri and tetra sialylated structures on glycans extracted from purified rFSH (Example 6) were measured using known techniques.

[0114] N Glycans were released from the samples using PNGase F under denaturative conditions and then were labeled with 2-aminobenzamide. Glycans were released from the samples using PNGase F under denaturative conditions and then labeled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of sialylation distribution. The relative amounts of neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated structures are shown in FIG. 5 (for the 8 samples shown in FIG. 4).

[0115] The rFSH includes neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated glycan structures with relative amounts as follows: neutral 5-6%; 15-17% mono-sialylated; 26-30% di-sialylated; 30-32% tri-sialylated and 17-23% tetra-sialylated.

Example 8a

[0116] The relative percentage amounts of α2,6 sialic acid on purified rFSH extracted from nine samples of purified rFSH (produced by the methods of Example 6) were measured using known techniques.

[0117] N-Glycans were released from the samples using PNGase F under denaturative conditions and then labelled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of charge distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column (see Example 8). The analysis allows calculation of α2,6 sialic acid.

[0118] The relative percentages of the charged glycans were calculated from structures present in the undigested and digested glycan pools and are shown in the following Table. These were found to be in the ranges 25 to 50%, generally 30 to 35% for α2,6 sialylation.

[0119] The relative percentage amounts of bisecting GlcNac, GalNac and 1-Fucose Lewis on glycans extracted from the nine samples of purified rFSH (produced by the methods of Example 6) were measured using known techniques. N-Glycans were released from the glycoprotein using PNGase F and labeled with 2-aminobenzamide (2AB). The analysis was done by two dimensional (2D) HPLC analysis in combination with enzymatic degradation of the glycans. For verification, the glycans were analyzed by MALDI-MS The relative amounts of alpha 2,6-sialic acid and the terminal residues are shown in the following table, together with those for Gonal F (CHO cell derived recombinant FSH) and Bravelle (human urinary FSH).

TABLE-US-00004 Ref. Ref. Gonal O N I-1 I-2 I-3 II II III-1 III-2 Average F Bravelle abun- abun- abun- abun- abun- abun- abun- abun- abun- abun- abun- abun- dance dance dance dance dance dance dance dance dance dance dance dance Sample % % % % % % % % % % % % 2,6 27.7 34.9 26.2 30.1 31.1 28.3 30.4 35 33 30.7 0 55.4 sialic acid 1GalNAc 51 44.6 50.7 44.7 49 47.6 45.3 46.4 44.9 47.1 0 11.3 Bisecting 10 12.4 10.2 8.9 8.7 11.8 11.4 10.6 13.9 10.9 55 14 GlcNAc 1 21.1 16.7 23.3 16.1 20.3 18.1 17.9 18.7 19.0 19.0 3.1.sup.1 2.2 Fucose Lewis 2 4 4.1 4.3 1.9 3.1 4.2 3.8 3.9 4.4 3.7 — n.d..sup.2 Fucose Lewis .sup.1Value of 3.1 is total 12 Fucose Lewis. .sup.2Not determined.

[0120] It can be seen that the amount of GalNac in the FSH of the invention varies between about 44.9 and 51%, averaging about 47.1%.

[0121] It can be seen that the amount of bisecting GlcNac in the FSH of the invention varies between 8.7 and 13.9%, averaging approximately at 10.9%.

[0122] It can be seen that the amount of 1 Fucose Lewis in the FSH of the invention varies between 16.1 and 23.3%, averaging approximately at 19%.

[0123] It can be seen that the amount of 2 Fucose Lewis in the FSH of the invention varies between 1.9 and 4.4%, averaging approximately at 3.7%.

Example 9—a Multiple Dose Study Investigating the Safety, Tolerability, Pharmacokinetics, Pharmacodynamics, and Immunogenicity of FE 999049 in Comparison to GONAL-F

[0124] Study Population

[0125] A total of 48 (24 on each drug) healthy women received daily doses of 14.6 μg of FE 999049 (a composition according to the invention, produced according to Example 6) or 16.5 μg of Gonal-F for seven days.

[0126] Safety Results

[0127] Multiple dose administration of FE 999049 and GONAL-F was safe and generally well tolerated as assessed by Adverse Events (AEs), vital signs, ECG, clinical laboratory measurements, and physical examination. No serious adverse event or death occurred during the study.

[0128] Pharmacokinetic Results

[0129] Following the administration of FE 999049 and GONAL-F over 7 days, the FSH concentration values as assessed immediately prior to the next injection increased and seemed to reach a steady state level after 6-7 days. However the exposure (AUC and Cmax) of FE 999049 was 60% higher in comparison to Gonal-F.

[0130] Pharmacodynamic Results

[0131] The concentrations of inhibin-B (see FIG. 6), oestradiol, and progesterone all increased subsequent to administration of FE 999049 and GONAL-F, however to a greater extent following administration of FE 999049 compared to GONAL-F. Both number and size distribution of follicles showed a greater response to FE 999049 compared to GONAL-F.

[0132] Example 9 demonstrates that FSH having a specific amount (17-23%) of tetra-sialylated glycan structures and e.g. specific amounts of α2,3 sialylation and α2,6 sialylation is markedly more potent then recombinant FSH products which are currently on the market.

Example 10—A Multiple Dose Study Investigating FE 999049 in Comparison to GONAL-F

[0133] The following describes a randomised, controlled, assessor-blind, parallel groups, multinational, multicentre trial assessing the dose-response relationship of FE 999049 in patients undergoing controlled ovarian stimulation for in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI). The patient population was 265 IVF patients aged between 18 to 37 years, with BMI 18.5 to 32.0 kg/m2.

[0134] The trial was designed as a dose-response trial with number of oocytes retrieved as the primary endpoint. Secondary endpoints will explore the qualitative and quantitative impact of different doses of FE 999049 with regard to endocrine profile, follicular development, oocyte fertilisation, embryo quality and treatment efficiency (i.e. total gonadotropin consumption and duration of stimulation). The trial is designed to evaluate the efficacy of FE 999049 to establish pregnancy when used in controlled ovarian stimulation for IVF/ICSI cycles.

[0135] Subjects were assessed within 3 months prior to randomisation for compliance with the inclusion and exclusion criteria, including an anti-Müllerian hormone (AMH) assessment to increase homogeneity of the trial population in relation to ovarian response and minimise the number of potential poor and hyper-responders to the FE 999049 doses and GONAL-F dose used in the trial. The AMH assessment was measured using the AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, Tex.). This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L.

[0136] On day 2-3 of their menstrual cycle, subjects were randomised in a 1:1:1:1:1:1 fashion to treatment with either 90 IU, 120 IU, 150 IU, 180 IU or 210 IU FE 999049 or 150 IU GONAL-F, and ovarian stimulation initiated. Randomisation was stratified according to AMH level at screening [5.0-14.9 pmol/L (low AMH) and 15.0 to 44.9 pmol/L (high AMH)).

[0137] Gonal-F is filled by mass (FbM) at FDA request; referring to μg dose is therefore appropriate. The Gonal-F label indicates 600 IU/44 μg, which indicates that 150 IU is 11 μg. However, there is some variation and the batch certificate for this trial indicated that 11.3 μg Gonal-F was equivalent to 150 IU. The FE999049 doses are presented by protein content (μg) rather than biological activity. Thus the doses of FE999049 were 5.2 μg (90 IU), 6.9 μg (120 IU), 8.6 μg (150 IU), 10.3 μg (180 IU) or 12.1 μg (210 IU).

[0138] The subject and dose distribution is set out as follows (data are number of subjects):

TABLE-US-00005 TABLE 1 5.2 6.9 FE 999049 10.3 12.1 GONAL-F μg μg 8.6 μg μg μg 11.3 (11) μg Total Screened 334 Randomised 42 45 44 45 46 43 265 and exposed High AMH 23 26 24 24 26 25 148 strata (56%) (15.0-44.9 pmol/L) Low AMH 19 19 20 20 21 18 117 strata (44%) (5.0-14.9 pmol/L) Per-protocol 40 42 42 44 44 43 255

[0139] The daily dose level of FE 999049 or GONAL-F is fixed throughout the entire stimulation period. During stimulation, subjects are monitored on stimulation day 1, 4 and 6 and hereafter at least every second day. When 3 follicles of ≥15 mm are observed, visits are performed daily. Subjects are treated with FE 999049 or GONAL-F for a maximum of 16 days.

[0140] To prevent a premature LH surge, a GnRH antagonist (ganirelix acetate, ORGALUTRAN, MSD/Schering-Plough) may be initiated on stimulation day 6 at a daily dose of 0.25 mg and continued throughout the stimulation period. Triggering of final follicular maturation is done on the day when ≥3 follicles with a diameter ≥17 mm are observed. If there are <25 follicles with a diameter ≥12 mm, 250 μg recombinant hCG (choriogonadotropin alfa, OVITRELLE, Merck Serono/EMD Serono) is administered. If there are 25-35 follicles with a diameter ≥12 mm, 0.2 mg GnRH agonist (triptorelin acetate, DECAPEPTYL/GONAPEPTYL, Ferring Pharmaceuticals) is administered. In case of excessive ovarian response, defined as >35 follicles with a diameter ≥12 mm, the treatment is cancelled. In case of poor ovarian response, defined as <3 follicles with a diameter ≥10 mm observed on stimulation day 10, the cycle could be cancelled.

[0141] Oocyte retrieval takes place 36 h (±2 h) after triggering of final follicular maturation and the oocytes inseminated by IVF and/or ICSI. Fertilisation and embryo development are assessed from oocyte retrieval to the day of transfer. For subjects who underwent triggering of final follicular maturation with hCG, one blastocyst of the best quality available is transferred on day 5 after oocyte retrieval while remaining blastocysts are frozen. For subjects who undergo triggering of final follicular maturation with GnRH agonist, no embryo transfer takes place in the fresh cycle and blastocysts are instead frozen on day 5. Vaginal progesterone tablets (LUTINUS, Ferring Pharmaceuticals) 100 mg 3 times daily are provided for luteal phase support from the day after oocyte retrieval until the day of the clinical pregnancy visit. A phCG test is performed 13-15 days after embryo transfer and clinical pregnancy will be confirmed by transvaginal ultrasound (TVU) 5-6 weeks after embryo transfer.

[0142] Results

[0143] The number of oocytes retrieved (primary endpoint) is shown in the following Table.

TABLE-US-00006 TABLE 2 FE 999049 GONAL-F 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg 11.3 (11) μg Oocytes retrieved All 5.2 (3.3) 7.9 (5.9)  9.2 (4.6) 10.6 (7.0) 12.2 (5.9) 10.4 (5.2) High AMH 5.9 (3.9) 9.1 (6.4) 10.6 (4.8) 13.6 (7.8) 14.4 (5.8) 12.4 (5.4) Low AMH 4.5 (2.2) 6.3 (4.9)  7.4 (3.8)  6.9 (3.6)  9.4 (4.9)  7.8 (3.4) Data are mean (SD)

[0144] The primary objective was met: a significant dose-response relationship was established for FE 999049 with respect to number of oocytes retrieved. This finding was observed not only for the overall trial population, but also for each of the two AMH strata used at randomisation.

[0145] A significant dose-response for FE 999049 was demonstrated for all key objective pharmacodynamic parameters, e.g. estradiol, inhibin B and inhibin A. At a similar microgram dose level, the pharmacodynamic responses with FE 999049 were larger than with GONAL-F (these results not shown).

[0146] The serum FSH concentrations after exposure to FE 999049 were significantly higher than for GONAL-F. The results confirm that the PK profile of FE 999049 differs from that of GONAL-F.

[0147] Fertilisation rates, blastocyst development and pregnancy rates in IVF/ICSI patients treated with FE 999049 were within expectations.

[0148] There were no safety concerns with the use of FE 999049. A good local tolerability was documented.

[0149] Further Analysis

[0150] The applicants have further analysed the data to identify the FE 999049 dose(s) that fulfil the following criteria with respect to number of oocytes retrieved: [0151] Oocytes retrieved in the range 8-14 [0152] Minimise proportion of patients with <8 oocytes [0153] Minimise proportion of patients with ≥20 oocytes

[0154] The applicants also investigated the impact of body weight. If relevant, the dose is converted into μg/kg for an average subject. This value of μg/kg and ±0.01 μg/kg are evaluated in a model with respect to distribution of oocytes retrieved as well as safety profile, and the optimal dose is identified.

[0155] Low AMH Strata

[0156] As seen in Table 2, the dose of FE999049 which fulfilled the first criterion (Oocytes retrieved in the range 8-14) was 12.1 μg (mean 9.4 oocytes retrieved). The distribution of oocytes is shown in Table 3 below.

TABLE-US-00007 TABLE 3 5.2 6.9 FE 999049 10.3 12.1 GONAL-F μg μg 8.6 μg μg μg 11.3 (11) μg Oocytes retrieved <4 32% 24% 15% 10% 10%  6% 4-7 63% 42% 45% 60% 20% 56%  8-14  5% 24% 35% 30% 60% 33% 15-19  0%  5%  5%  0%  5%  6% ≥20  0%  5%  0%  0%  5%  0% Data are % of subjects

[0157] As shown by the arrow, a dose of 12.1 μg FE999049 provides retrieval of the most desirable number of oocytes in 60% of subjects in the low AMH group. This is a marked improvement on Gonal-F (most desirable number of oocytes in only 33% of subjects).

[0158] Table 4 below shows the analysis of signs of excessive response in the low AMH strata (data are number of subjects). It can be seen that there were no indications of early OHSS of a moderate or severe nature and there were no incidences of preventative action being required; there are no concerns associated with the dose of 12.1 μg FE999049 in a patient having low AMH.

TABLE-US-00008 TABLE 4 FE 999049 GONAL-F 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg 11.3 (11) μg All subjects 19 19 20 20 21 18 Early OHSS, mod/sev 0 0 0 0 0 0 GnRH agonist triggering 0 0 0 0 0 0 Preventive action* 0 0 0 0 0 0 ≥15 oocytes 0 2 1 0 2 1 Any of the above 0 2 1 0 2 1

[0159] FIG. 7 shows the effect of body weight on oocytes retrieved (for the low AMH strata), for the various doses. The arrows indicate the number of oocytes retrieved from subjects of bodyweight between 45 kg and 90 kg treated at the 12.1 μg dose. As can be seen the variation between patients of bodyweight 45 kg and those of 90 kg is less than around 0.5 oocytes; in other words dosing by body weight is not required in patients with low AMH when dose of FE999049 is at least 12 μg, because there is not a significant variation in oocytes retrieved with body weight, at this dose.

[0160] Thus the applicants have found that a dose of, or dose equivalent to, 6 to 18 μg, for example 9 to 14 μg, for example 12 μg, human derived recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH<15 pmol/L, for example 0.05-14.9 pmol/L for example 5.0-14.9 pmol/L. The dose provides an effective response while minimising risk of OHSS.

[0161] High AMH Strata

[0162] As seen in Table 2, three doses of FE999049 fulfilled the first criterion (oocytes retrieved in the range 8-14): 6.9 μg (mean 9.1 oocytes retrieved), 8.6 μg (mean 10.6 oocytes retrieved), and 10.3 μg (mean 13.6 oocytes retrieved).

[0163] FIG. 8 shows the effect of body weight on oocytes retrieved (for the high AMH strata), for the various doses. The arrows indicate the number of oocytes retrieved from subjects of body weight between 45 kg and 90 kg treated at the 6.9 μg, 8.6 μg and 10.3 μg doses. As can be seen the variation is significant: for the 6.9 μg dose 6 additional oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient; for the 8.6 μg dose 4 additional oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient; and for the 10.1 μg dose 2.5 additional oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient. In other words dosing by body weight has an impact in patients with high AMH when the dose of FE999049 is less than 12 μg, because there is a significant variation in oocytes retrieved with body weight, at these doses.

[0164] Table 5a below shows a further breakdown of oocytes retrieved (from Table 2) by AMH. This shows the doses which fulfilled the first criterion (oocytes retrieved in the range 8-14) for each sub strata of AMH.

TABLE-US-00009 TABLE 5a FE 999049 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg Oocytes retrieved 15-24 pmol/L 4.9 7.3 10.6 11.5 12.3 (3.8) (3.6) (5.1) (6.7) (5.9) 25-34 pmol/L 7.0 9.1 9.7 15.5 16.7 (1.8) (6.8) (6.7) (6.4) (4.9) 35-45 pmol/L 8.5 21.0 11.3 18.0 15.7 (9.2) (1.4) (2.6) (12.2) (6.5)

[0165] Table 5 b below shows the analysis of patients where treatment was cancelled due to either excessive response or agonist triggering, for these subgroups. For example, one patient in the 25-34 pmol/L AMH strata cancelled due to excessive response following the dose of 10.3 μg and one patient in the 25-34 pmol/L AMH strata cancelled due to excessive response following the dose of 12.1 μg; one patient in the 35-45 pmol/L AMH strata cancelled following agonist triggering following dose of 10.3 μg; and one patient in the 35-45 pmol/L AMH strata cancelled following agonist triggering following dose of 6.9 μg.

TABLE-US-00010 TABLE 5b FE 999049 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg OHSS***, cancellation due to excessive response or agonist triggering**** 15-24 pmol/L 0 0      0 0     0     25-34 pmol/L 0 0      0 1*** 1*** 35-45 pmol/L 0 1**** 0   1**** 0    

[0166] It can be seen therefore that tailoring of dose by bodyweight (FIG. 8) and AMH level would be useful in the high AMH strata, to minimise cancellations and maximise oocyte retrieval.

[0167] The applicants have found that the following doses provide an effective response while minimising risk of OHSS (kg is kg body weight of patient).

TABLE-US-00011 Serum AMH dose (Max dose)   <15 pmol/L 12 μg (12 μg) 15-24 pmol/L 0.14-0.19 μg/kg, (12 μg) for example 0.15-0.16 μg/kg, preferably 0.15 μg/kg 25-34 pmol/L 0.11-0.14 μg/kg; (12 μg) for example 0.12-0.13 μg/kg, preferably 0.13 μg/kg   ≥35 pmol/L 0.10-0.11 μg/kg, (12 μg) preferably 0.11 μg/kg

[0168] The following are appropriate if dosing by bodyweight is not desired.

TABLE-US-00012 Serum AMH dose (Max dose)   <15 pmol/L   12 μg 12 μg 15-24 pmol/L 9.3-10 μg  (12 μg) 25-34 pmol/L 7.3-8 μg (12 μg)   ≥35 pmol/L 6.3-7 μg (12 μg)

[0169] The following are appropriate if fewer categories of AMH are required.

TABLE-US-00013 4 AMH 3 AMH 2 AMH categories categories categories One dose AMH Dose AMH Dose AMH Dose AMH Dose <15 12 μg <15 12 μg <15 12 μg — 0.16 μg/kg 15-24 0.15-0.16 15-24 0.15-0.16 ≥15 0.14 μg/kg μg/kg μg/kg 25-34 0.12-0.13 ≥25 0.12 μg/kg μg/kg ≥35 0.10-0.11 μg/kg

[0170] The following are appropriate if dosing by bodyweight is not desired.

TABLE-US-00014 4 AMH 3 AMH 2 AMH categories categories categories One dose AMH Dose AMH Dose AMH Dose AMH Dose <15 12 μg <15  12 μg <15  12 μg — 9.3 μg or 10 15-24 9.3-10 μg 15-24 9.3-10 μg ≥15 8.7 μg μg 25-34 7.3-8 μg ≥25 7.3 μg ≥35 6.3-7 μg

[0171] Thus the applicants have found that a dose of, or dose equivalent to, 9 to 14 μg, for example 12 μg, human derived recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH<15 pmol/L, for example 0.05-14.9 pmol/L for example 5.0-14.9 pmol/L. The dose provides an effective response while minimising risk of OHSS.

[0172] The applicants have found that a dose of, or dose equivalent to, 5 to 12.5 μg, for example 6 to 10.5 μg, human derived recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH>15 pmol/L. The dose provides an effective response while minimising risk of OHSS.

[0173] The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.09 to 0.19 μg human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of ≥15 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.14 to 0.19 μg human derived recombinant FSH (preferably 0.15 to 0.16 μg human derived recombinant FSH) per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 15 to 24.9 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.11 to 0.14 μg human derived recombinant FSH (preferably 0.12 to 0.13 μg human derived recombinant FSH) per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 25 to 34.9 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.10 to 0.11 μg human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 35 pmol/L. These doses provide an effective response while minimising risk of OHSS.

[0174] The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 μg (e.g. 0.16 μg) human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of <15 pmol/L, for example for the first stimulation cycle with human derived recombinant FSH. However, it is not required that patients are dosed by body weight at this level of AMH.

Example 10 A—Individualised COS Protocol (Low AMH)

[0175] The selected patients are about to undergo COS for in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) by methods known in the art. The pre-treatment protocol includes assessment/screening of the patient's serum AMH using the AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, Tex.). This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L. AMH may be measured using other Assay kits (e.g. available from Roche).

[0176] The COS protocol proceeds in the usual manner apart from administration of the initial dose of FE 999049 according to AMH level at screening. A patient with an AMH level of <14.9 pmol/L would be administered an initial daily dose of approximately 12 μg FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 15 to 24.9 pmol/L would receive an initial daily dose of 0.15 to 0.19 μg of the human derived recombinant FSH per kg bodyweight of the patient. A patient with an AMH level of 25 to 34.9 pmol/L would receive an initial daily dose of 0.11 to 0.13 μg of the human derived recombinant FSH per kg bodyweight of the patient. A patient with an AMH level of 35 pmol/L would receive an initial daily dose of 0.10 to 0.11 μg of the human derived recombinant FSH per kg bodyweight of the patient.

Example 11—Individualised COS Protocols

[0177] The doses in this protocol are less preferred that Example 10A.

[0178] The selected patients are about to undergo COS for in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) by methods known in the art. The pre-treatment protocol includes assessment/screening of the patient's serum AMH using the AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, Tex.). This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L.

[0179] The COS protocol proceeds in the usual manner apart from administration of the initial dose of FE 999049 according to AMH level at screening in line with the following table. Thus a patient with an AMH level of 5-14.8 pmol/L would be administered 180 IU FSH in the form of approximately 8-11 μg FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 30-44.9 pmol/L would be administered 120 IU FSH in the form of approximately 4-7 μg FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6. If the AMH level is not available, the patient recombinant would be administered 120-180 IU FSH in the form of approximately 6-11 μg FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6.

TABLE-US-00015 Starting Dose Approx equivalent AMH level FSH in μg      <5 pmol/l 210 IU 10-15 μg     5-14.9 pmol/l 180 IU 8-11 μg  >15-29.9 pmol/l 150 IU 6-9 μg >30-44.9 pmol/l 120 IU 4-7 μg     >45 pmol/l  90 IU 2-5 μg Not Available 120-180 IU 6-11 μg 

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TABLE-US-00016 Follicle stimulating hormone alpha polypeptide Accession number AH007338 Nucleotide sequence of FSH alpha SEQ ID NO: 1 1 ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT 61 GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA 121 TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA 181 TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG 241 TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG 301 GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA A Protein sequence of FSH alpha (SEQ ID NO: 5) 1 MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QCMGCCFSRA 61 YPTPLRSKKT MLVQKNVTSE STCCVAKSYN RVTVMGGFKV ENHTACHCST CYYHKS Follicle stimulating hormone beta polypeptide Accession number NM_000510 Nucleotide sequence of FSH beta SEQ ID NO: 2 1 ATGAAGACAC TCCAGTTTTT CTTCCTTTTC TGTTGCTGGA AAGCAATCTG CTGCAATAGC 61 TGTGAGCTGA CCAACATCAC CATTGCAATA GAGAAAGAAG AATGTCGTTT CTGCATAAGC 121 ATCAACACCA CTTGGTGTGC TGGCTACTGC TACACCAGGG ATCTGGTGTA TAAGGACCCA 181 GCCAGGCCCA AAATCCAGAA AACATGTACC TTCAAGGAAC TGGTATATGA AACAGTGAGA 241 GTGCCCGGCT GTGCTCACCA TGCAGATTCC TTGTATACAT ACCCAGTGGC CACCCAGTGT 301 CACTGTGGCA AGTGTGACAG CGACAGCACT GATTGTACTG TGCGAGGCCT GGGGCCCAGC 361 TACTGCTCCT TTGGTGAAAT GAAAGAATAA Protein sequence of FSH beta (SEQ ID NO: 6) 1 MKTLQFFFLF CCWKAICCNS CELTNITIAI EKEECRFCIS INTTWCAGYC YTRDLVYKDP 61 ARPKIQKTCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS 121 YCSFGEMKE Beta-galactoside alpha-2,3-sialyltransferase 4 Accession Number L23767 Nucleotide sequence of ST3GAL4 SEQ ID NO: 3 1 ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG 61 TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG 121 AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC 181 TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA 241 TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA 301 GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG 361 GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC 421 GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC 481 AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC 541 AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG 601 ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC 661 TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT 721 GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG 781 GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT 841 GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC 901 AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG 961 CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA Protein Sequence of ST3GAL4 (SEQ ID NO: 7) 1 MCPAGWKLLA MLALVLVVMV WYSISREDRY IELFYFPIPE KKEPCLQGEA ESKASKLFGN 61 YSRDQPIFLR LEDYFWVKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV 121 VVGNGHRLRN SSLGDAINKY DVVIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN 181 NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA 241 DKLLSLPMQQ PRKIKQKPTT GLLAITLALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL 301 KSMAGSGHNV SQEALAIKRM LEMGAIKNLT SF Beta-galactosamide alpha-2,6-sialyltransferase 1 Accession number NM_003032 Nucleotide sequence of ST6GAL1 SEQ ID NO: 4 1 ATGATTCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT 61 GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG 121 CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC 181 CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT 241 CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC 301 TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC 361 AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG 421 CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC 481 AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT 541 TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA 601 GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA 661 CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG 721 AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGGAATCC TAATTGTATG GGACCCATCT 781 GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC 841 TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG 901 CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC 961 CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT 1021 TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC 1081 GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA TTTGGTGAAG 1141 CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC 1201 TTCCGGACCA TTCACTGCTA A 0p-Protein Sequence of ST6GAL1 (SEQ ID NO: 8) 1 MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS 61 QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN 121 KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP 181 WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE 241 KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM 301 PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF 361 DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC