Enhancing the production of Anti-mullerian hormone through modification of its prodomain

Abstract

In one aspect of the present invention, a genetically modified Anti-Mullerian Hormone (AMH) is disclosed where one or more cysteine residues in the prodomain are replaced with amino acid residues other than cysteine. In one embodiment, one or more cysteine residues in the prodomain are replaced with serine residues. In one embodiment, cysteine 241 of the prodomain is replaced with a serine residue.

Description

ENHANCING THE PRODUCTION OF ANTI-MUELERIANHORMONE THROUGH MODIFICATION OF ITS PRODOMAINCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 535,395, filed August 30, 2023, which application is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] This invention relates generally to modifications of prodomains.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under HD105818 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0004] This section is intended to introduce the reader to various aspects of the art that may be related to various aspects of the present invention, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0005] Anti-Mullerian Hormone (AMH) is a member of the transforming growth factor beta (TGFP) family of signaling ligands. AMH is essential during male development of the reproductive axis where it drives the regression of the Mullerian duct, a structure that, in females, develops into the female reproductive tract. Postnatally, AMH plays a role in females where it can suppress the follicle selection and maturation. Therefore, there is a clinical need to modulate AMH activity in order to have better control over the rate of ovulation in the female. For example, one thought is that AMH can be used during oncofertility where women undergo chemotherapy and would like to preserve their reproductive health. In animal models, AMH was shown to halt folliculogenesis during chemotherapy. Subsequently, once thechemotherapy was completed, AMH was removed, and the animals had an increased chance of live births.

[0006] As a member of the TGF[3 family, the AMH gene consists of a signal sequence, a prodomain, a furin cleavage recognition site and the mature component. The signal sequence allows for secretion, the prodomain allows for proper folding of the mature component, the furin allows for proteolysis of the prodomain from the mature component. The final signal molecule is a disulfide linked dimer of two mature components. After cleavage the prodomain remains noncovalently bound to the mature termed a procomplex (prodomain + mature). In a biological setting, the procomplex exhibits much more activity than the mature component alone. Thus, current strategies include the procomplex during treatment.

[0007] Recombinant protein production is complicated in that over-expression of AMH results in several disulfide-bonded aggregates. This complicates production and results in a heterogenous population of AMH procomplex. These aggregates disassemble when reduced demonstrating that they are disulfide bonded. The prodomain contains 6 cysteine residues which are thought to be involved in the aggregation of the protein. Like the mature, the prodomain is also disulfide-linked through an intermolecular bond.SUMMARY OF THE INVENTION

[0008] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0009] In one aspect of the present invention, a genetically modified Anti-Mullerian Hormone (AMH) is disclosed where one or more cysteine residues in the prodomain are replaced with amino acid residues other than cysteine. In one embodiment, one or more cysteine residues in the prodomain are replaced with serine residues. In one embodiment, cysteine 241 of the prodomain is replaced with a serine residue. In another embodiment, cysteine 55 of the prodomain is replaced with a serine residue.

[0010] In another aspect of the present invention, a genetically modified transforming growth factor beta (TGF[3) protein is disclosed where one or more cysteine residues in the prodomain are replaced with amino acid residues other than cysteine. In one embodiment, one or more cysteine residues in the prodomain are replaced with serine residues. In one embodiment, the protein is Bone Morphogenetic Protein 3 (BMP3) and cysteine 272 of theprodomain is replaced with a serine residue. In another embodiment, the protein is Bone Morphogenetic Protein 6 (BMP6) and a cysteine selected from the group consisting of C21, C22 and C412 of the prodomain is replaced with a serine residue.

[0011] In one embodiment, the protein is Bone Morphogenetic Protein 8 A (BMP8A) and a cysteine selected from the group consisting of C31 and C205 of the prodomain is replaced with a serine residue. In another embodiment, the protein is Bone Morphogenetic Protein 8B (BMP8B) and a cysteine selected from the group consisting of C31 and C205 of the prodomain is replaced with a serine residue. In one embodiment, the protein is Growth Differentiation Factor 10 (GDF10) and cysteine 150 of the prodomain is replaced with a serine residue. In another embodiment, the protein is Growth Differentiation Factor 3 (GDF3) and cysteine 227 of the prodomain is replaced with a serine residue. In one embodiment, the protein is Growth Differentiation Factor 8 (GDF8) and a cysteine selected from the group consisting of C39 and C42 of the prodomain is replaced with a serine residue.

[0012] In another embodiment, the protein is Growth Differentiation Factor 9 (GDF9) and a cysteine selected from the group consisting of C120 and C176 of the prodomain is replaced with a serine residue. In one embodiment, the protein is Activin C (ActC) and a cysteine selected from the group consisting of C96 and Cl 90 of the prodomain is replaced with a serine residue. In another embodiment, the protein is Activin E (ActE) and a cysteine selected from the group consisting of C139 and C192 of the prodomain is replaced with a serine residue. In one embodiment, the protein is TGFpi and cysteine 33 of the prodomain is replaced with a serine residue. In another embodiment, the protein is TGFP2 and a cysteine selected from the group consisting of C24 and C89 of the prodomain is replaced with a serine residue. In one embodiment, the protein is TGFP3 and a cysteine selected from the group consisting of C27, C91 and C123 of the prodomain is replaced with a serine residue.

[0013] In another aspect of the present invention, a method of inducing contraception in an animal is disclosed. The method involves administering one or more doses of the genetically modified AMH of claim 1 to the animal. In one embodiment, the animal is a cat. In another embodiment, the animal is a dog.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The objects and advantages of the present invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0015] FIG. 1A is a graph showing a cell-based signaling assay.

[0016] FIG. IB is an image showing Mullerian duct regression in rat urogenital ridge with and without prodomain.

[0017] FIG. 2A is a schematic showing an embodiment of disulfide patterning within WT AMH according to the present invention. S200: 150kDa, NR: 1 lOkDa, R: 55kDa.

[0018] FIG. 2B is a schematic showing an oligomer according to the present invention driven by cys241. S200:300kDa, NR: 250kDa, R: 55kDa.

[0019] FIG. 2C is a schematic showing an oligomer according to the present invention driven by cys241. S200:300kDa, NR: lOkDa, R: 55kDa.

[0020] FIG. 3 is a schematic showing a model of AMH Prodomain.

[0021] FIG. 4 is a schematic and graph showing cys mutation expression data.

[0022] FIG. 5 is a graph showing that mutant AMH constructs according to the present invention produce active protein.

[0023] FIG. 6 is a graph showing increased expression and decreased aggregation (+homogeneity) of cys mutants according to the present invention.

[0024] FIG. 7A is a pair of images of westerns for nonreduced and reduced.

[0025] FIG. 7B is a graph showing 50mL conditioned media of prodomain only.

[0026] FIG. 8A is a pair of images of westerns for nonreduced and reduced

[0027] FIG. 8B is a graph showing pooling around the A8 peak.

[0028] FIG. 9 is a graph showing an overlay of sizing peaks.

[0029] FIG. 10 is an assay showing expression and function of LR-MIS cys mutants in vitro.

[0030] FIG. 11 A is an image from the gold-standard bioassay for AMH function for PA5- 5ug / ml.

[0031] FIG. 1 IB is an image from the gold-standard bioassay for AMH function for PA5- 25ug / ml.

[0032] FIG. 11C is an image from the gold-standard bioassay for AMH function for QB9-5ug / ml.

[0033] FIG. 1 ID is an image from the gold-standard bioassay for AMH function for QB9-5ug / ml.

[0034] FIG. 12 is an assay showing conditioned media signaling activity.DEFINITIONS

[0035] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration, or percentage, is meant to encompass variationsof, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods.

[0036] As used herein, “amino acid residues other than cysteine” includes histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, glutamic acid, serine, arginine, glutamine, glycine, proline, and tyrosine.DETAILED DESCRIPTION OF THE INVENTION

[0037] One or more specific embodiments of the present invention are described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not necessarily be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0038] In one embodiment, the present invention involves the use of AMH analogs with modified cysteine residues in the prodomain to increase recombinant production, reduce aggregation, and produce a more homogenous population of AMH. Modifications of cysteine residues within the Anti-Mullerian Hormone prodomain can be used in production for increased homogeneity, increased production, decreased aggregation and oligomerization, and an ability to reform a complex using exogenously produced and purified AMH prodomain.Anti-Mullerian Hormone (AMH) prodomain

[0039] Mammalian production of AMH requires a prodomain for secretion of mature signaling ligand. Prodomain is completely required for in vivo activity. All protein therapeutics utilizing the mature signaling component need to have a prodomain or a prodomain alternative present. While a cell-based signaling assay (FIG. 1A) shows similar activity with and without prodomain, an ex vivo assay for biological function (Mullerian duct regression in rat urogenital ridge) requires prodomain (FIG. IB).

[0040] Wild-type AMH (or current expression constructs which typically improve the leader sequence and the proteolytic cleave site between prodomain and mature AMH) have a number of current issues. One issue is the aggregation of produced protein leading to decreased yield. Another issue is that tetramer and higher order forms, which are active in signaling, but which are distinct molecules, create heterogeneity. Additionally, cysteine shuffling and reorganization over time and after freeze / thaw leads to further loss due to oligomerization and aggregation following initial purification. Finally, exogenously produced and purified wildtype prodomain cannot form a complex with mature AMH.Modified AMH

[0041] In one embodiment, the present invention involves AMH having mutations which replace cysteine residues within the prodomain with amino acid residues other than cysteine that improve the production, purity, and functionality of AMH as a clinical target. It has been surprisingly discovered that the mutation of cys241 to serine within the AMH prodomain does not affect activity while increasing overall production 3 -fold and decreasing aggregation 3- fold. The mutation of cys411 to serine allows for the formation of a complex between mature AMH and exogenously produced prodomain.

[0042] Functional data of AMH modified according to the present invention shows maintained signaling activity of mutations. Comparisons of single and combined cysteine mutations show structural differences and differences in expression and aggregation. The data shows no modification to the signaling component.

[0043] FIG. 2A is an embodiment of disulfide patterning within WT AMH according to the present invention. FIGs 2B and 2C show oligomers according to the present invention driven by cys241.

[0044] FIG. 3 shows the current model supported by evidence: internal structural disulfide between 103-188, critical for folding and expression. Disulfide between C55 of two prodomain chains forms homodimer. Compensatory / additional homodimer or oligomerization formed using available cys241. C411 is unbound but it will form a disulfide in the absence of mature AMH and prevent the prodomain from binding mature AMH.

[0045] FIG. 4A shows cys mutation expression data. Normalized western shown, raw western used for quantification.Protein production

[0046] Mutant AMH constructs according to the present invention produce active protein. Referring to FIG. 5, Y axis is signaling through a BRE reporter in fold activation relative to empty vector cell culture media (negative / normalization control for AMH expression media). X axis is volume of conditioned media added to reporter cells (20ul represents a 5-fold dilution of expression media). Cells transfected to respond to AMH signaling through AMHR2. All constructs produce functional and cleaved mature AMH at levels as high as or higher than WT.

[0047] FIG. 6 shows increased expression and decreased aggregation (+homogeneity) of cys mutants according to the present invention. Deletion of C241 decreases overall yield, but increases non-aggregated AMH by about two-fold. C241 S and C241 / 411 S have both increased total AMH and non-aggregated AMH production with C411S being the best target based on current data and our structural / biochemical data. Monomeric AMH (C55 / 241 / 411 S) has decreased overall expression but decreased aggregation (more homogenous).

[0048] The present invention may be expanded to other TGF[3 prodomains. Five models were generated of the noncovalent homodimer procomplex of each of 32 TGF-beta family ligands using Alphafold 2. Unpaired cysteines were identified which could increase aggregation and disrupt production. The proteins and cys residues of this analysis are shown in Table 1.Table 1

[0049] This would include single C-S mutations for each one as well as the combinatorial A,B,C,AB,AC,BC,ABC for multiple cys within a single protein.Recomplex

[0050] The present invention has found that prodomain C411S mutation allows exogenously produced prodomain to recomplex with mature AMH. FIG. 7 shows 50mL conditioned media of prodomain only C411 S from 4D expression, Expi293, PEI. FIG. 8 shows pooling around the A8 peak, concentrated to 500ul, sized on S200 (lOmMMES, 150mMNaCl, PH5).

[0051] FIG. 9 is a graph showing an overlay of sizing peaks. The solid line is a recomplex Pro C41 IS + Mature AMH. The dotted line is Pro only C41 IS. The data shows a leftward shift of about 0.2mL. From std curve =~20kDa shift estimated. AMH Mature does not size if not complexed.Non-surgical sterilization of animals

[0052] In another embodiment, the present invention can be used for non-surgical sterilization of animals such as dogs and cats. A study has found that a single dose of AMH gene therapy can induce long-term contraception in the domestic cat. The present invention allows for modifications to AMH so that more can be made from a smaller amount of virus. Additionally, this approach can be used for the non-surgical sterilization of dogs. The production of the dog gene is lOx less than cat.

[0053] Referring to FIGs 11A-11D, images from the gold-standard bioassay for AMH function are shown. 5 ug / mL concentration is the minimum of WT AMH that will be a full regression of the mullerian duct (pointed to by the white arrows.) The scale of 1 to 5 represents regression or bioactivity.

[0054] FIG. 11 A is an image of a bioassay for PA5-5ug / ml. PA5 indicates serine replacing Cys 411 in the prodomain. A 5 ug / mL concentration was used. FIG. 11B is an image of a bioassay for PA5-25ug / ml. A 25 ug / mL concentration was used. FIG. 11C is an image of a bioassay for QB9-5ug / ml. QB9 indicates a triple mutant, with serine replacing the cysteines C55S, C241 S, C411S in the prodomain. A 5 ug / mL concentration was used. FIG. 11D is an image of a bioassay for QB9-25ug / ml. A 25 ug / mL concentration was used. Overall scoring for each bioassay is as follows:

[0055] PA5, 5ug / mL - 4

[0056] PA5, 25ug / mL - 5

[0057] QB9, 5ug / mL - 4

[0058] QB9, 25ug / mL - largely ungradeable due to detachment of the ridge from muscle tissue over the course of the assay.

[0059] The results indicate that the variants maintained bioactivity in an in vivo context. The results of other bioassays are shown in Table 2.

[0060] Table 2: Bioactivity list for tested variants

[0061] Test materials had different identifiers in the examples. Table 3 shows the codes used for the same material. For example, “FL WT” was also referred to as “LH1” or “FL-A.”

[0062] Table 3

[0063] FIG. 10 is an assay showing expression and function of LR-MIS cys mutants in vitro. FIG. 12 is an assay showing conditioned media signaling activity.

[0064] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

What is claimed is:

1. A genetically modified Anti -Mullerian Hormone (AMH) having a prodomain wherein one or more cysteine residues in the prodomain are replaced with amino acid residues other than cysteine.

2. The AMH of claim 1 wherein cysteine 241 of the prodomain is replaced with a serine residue.

3. The AMH of claim 1 wherein cysteine 55 of the prodomain is replaced with a serine residue.

4. A genetically modified transforming growth factor beta (TGF0) protein having a prodomain wherein one or more cysteine residues in the prodomain are replaced with amino acid residues other than cysteine.

5. The TGFp protein of claim 4 wherein the protein is Bone Morphogenetic Protein 3 (BMP3) and cysteine 272 of the prodomain is replaced with a serine residue.

6. The TGFp protein of claim 4 wherein the protein is Bone Morphogenetic Protein 6 (BMP6) and a cysteine selected from the group consisting of C21, C22 and C412 of the prodomain is replaced with a serine residue.

7. The TGFp protein of claim 4 wherein the protein is Bone Morphogenetic Protein 8A (BMP8A) and a cysteine selected from the group consisting of C31 and C205 of the prodomain is replaced with a serine residue.

8. The TGFp protein of claim 4 wherein the protein is Bone Morphogenetic Protein 8B (BMP8B) and a cysteine selected from the group consisting of C31 and C205 of the prodomain is replaced with a serine residue.

9. The TGFp protein of claim 4 wherein the protein is Growth Differentiation Factor 10 (GDF10) and cysteine 150 of the prodomain is replaced with a serine residue.

10. The TGFp protein of claim 4 wherein the protein is Growth Differentiation Factor 3 (GDF3) and cysteine 227 of the prodomain is replaced with a serine residue.

11. The TGFp protein of claim 4 wherein the protein is Growth Differentiation Factor 8 (GDF8) and a cysteine selected from the group consisting of C39 and C42 of the prodomain is replaced with a serine residue.

12. The TGFp protein of claim 4 wherein the protein is Growth Differentiation Factor 9 (GDF9) and a cysteine selected from the group consisting of C120 and C176 of the prodomain is replaced with a serine residue.

13. The TGFp protein of claim 4 wherein the protein is Activin C (ActC) and a cysteine selected from the group consisting of C96 and Cl 90 of the prodomain is replaced with a serine residue.

14. The TGFp protein of claim 4 wherein the protein is Activin E (ActE) and a cysteine selected from the group consisting of C139 and C192 of the prodomain is replaced with a serine residue.

15. The TGFp protein of claim 4 wherein the protein is TGFpi and cysteine 33 of the prodomain is replaced with a serine residue.

16. The TGFp protein of claim 4 wherein the protein is TGFP2 and a cysteine selected from the group consisting of C24 and C89 of the prodomain is replaced with a serine residue.

17. The TGFP protein of claim 4 wherein the protein is TGFP3 and a cysteine selected from the group consisting of C27, C91 and C123 of the prodomain is replaced with a serine residue.

18. A method of inducing contraception in an animal comprising administering one or more doses of the genetically modified AMH of claim 1 to the animal.

19. The method of claim 18 wherein the animal is a cat.

20. The method of claim 18 wherein the animal is a dog.

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