EP-4735591-A2 - ENGINEERED ACID ALPHA-GLUCOSIDASE VARIANTS

EP4735591A2EP 4735591 A2EP4735591 A2EP 4735591A2EP-4735591-A2

Abstract

The present disclosure provides engineered acid alpha-glucosidase polypeptides, recombinant polynucleotides encoding the engineered acid alpha-glucosidase polypeptides, and method of using the engineered acid alpha-glucosidase polypeptides and the recombinant polynucleotides for therapeutic purposes.

Inventors

BOTHAM, Rachel, Cathleen
CHEUNG, Gordon, Michael
HALLOWS, William, Casey
HUISMAN, GJALT, W.
KRUSE, Nikki, D.
LAO, Jessica, P.
MAROJU, Shilpa
SILVERMAN, Adam, P.

Assignees

Crosswalk Therapeutics, Inc.

Dates

Publication Date: 20260506
Application Date: 20240628

Claims (1)

Docket No. CX7-223WO1 CLAIMS WHAT IS CLAIMED IS: 1. An engineered acid alpha-glucosidase, or biologically active fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more sequence identity to a reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises at least a substitution or amino acid residue 305V, 24A/C/D/F/G/H/I/K/M/N/P/S/T/V/Y, 28A/C/D/E/F/G/H/K/Q/T/V/W, 29A/C/D/E/F/G/H/I/K/M/N/P/R/W/Y, 39A/E/F/G/I/L/N/T, 50A/C/D/E/F/H/I/K/M/N/R/S/T/W/Y, 62D/H/I/K/M/N/P/Q/Y, 78A/C/D/F/G/H/I/K/L/M/N/Q/R/S/T/V/W/Y, 87A/G/H/I/K/L/MN/Q/R/S/T/V/W, 135C/D/E/F/G/H/I/K/L/N/R/Y, 266A/D/E/H/K/Q, 267H/L/T/V, 437A/H, 486C/D/F/G/H/I/K/L/M/N/Q/R/S/V/W/Y, 522A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/W/Y, 569A/C/D/E/G/K/M/N/P/R/W, 670A/D/G/H/K/M/Y, 692A/D/E/H/K/L/M/N/T/W, 711D/E/I/K/M/N/Q/S/T/V/Y, 736F/L, 750E/K/L/Q/R, 812A/D/G/S, 830D/E/F/G/H/L/M/N/S/T/W/Y, 842A/C/D/F/H/K/L/M/N/Q/R/T/W, 871A/C/D/F/H/I/M/N/Q/T/V/W/Y, 883A/F/Q, 894A/D/E/H/I/K/L/M/N/S/T/V/W/Y, 913F/I/K/M/N/S, or 932C/D/E/G/H/K/L/M/N/P/Q/R/W/Y, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 12 or 2, or to a reference sequence corresponding to SEQ ID NO: 12 or 2. 2. An engineered acid alpha-glucosidase, or biologically active fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-944 of an even numbered SEQ ID NO. of SEQ ID NOs: 14-754, or to an even numbered SEQ ID NO. of SEQ ID NOs: 14-754, wherein the amino acid sequence comprises at least a substitution or amino acid residue 305V, 24A/C/D/F/G/H/I/K/M/N/P/S/T/V/Y, 28A/C/D/E/F/G/H/K/Q/T/V/W, 29A/C/D/E/F/G/H/I/K/M/N/P/R/W/Y, 39A/E/F/G/I/L/N/T, 50A/C/D/E/F/H/I/K/M/N/R/S/T/W/Y, 62D/H/I/K/M/N/P/Q/Y, 78A/C/D/F/G/H/I/K/L/M/N/Q/R/S/T/V/W/Y, 87A/G/H/I/K/L/MN/Q/R/S/T/V/W, 135C/D/E/F/G/H/I/K/L/N/R/Y, 266A/D/E/H/K/Q, 267H/L/T/V, 437A/H, 486C/D/F/G/H/I/K/L/M/N/Q/R/S/V/W/Y, 522A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/W/Y, 569A/C/D/E/G/K/M/N/P/R/W, 670A/D/G/H/K/M/Y, 692A/D/E/H/K/L/M/N/T/W, 711D/E/I/K/M/N/Q/S/T/V/Y, 736F/L, 750E/K/L/Q/R, 812A/D/G/S, 830D/E/F/G/H/L/M/N/S/T/W/Y, 842A/C/D/F/H/K/L/M/N/Q/R/T/W, 871A/C/D/F/H/I/M/N/Q/T/V/W/Y, 883A/F/Q, 894A/D/E/H/I/K/L/M/N/S/T/V/W/Y, 913F/I/K/M/N/S, or 932C/D/E/G/H/K/L/M/N/P/Q/R/W/Y, or Docket No. CX7-223WO1 combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 20 to 944 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2. 3. The engineered acid alpha-glucosidase of claim 1 or 2, wherein the amino acid sequence of the engineered acid alpha-glucosidase comprises residues 20 to 944 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-754, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 14-754. 4. The engineered acid alpha-glucosidase of claim 1 or 2, wherein the amino acid sequence of the engineered acid alpha-glucosidase comprises residues 20 to 944 of SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506, or comprises SEQ ID NO: 14, 114, 126, 170, 250, 252, 394, 472, 488, or 506. 5. The engineered acid alpha-glucosidase of any one of claims 1-4, wherein the engineered acid alpha-glucosidase exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to pH 7; iii) increased tolerance to pH 4.4; iv) increased stability in lysosomes; v) increased expression in cells; vi) increased uptake into cells; vii) increased enzymatic activity in cell lysates; viii) increased stability in plasma/serum; and ix) reduced immunogenicity; or a combination of any of i), ii), iii), iv), v), vi), vii), viii), and ix), as compared to a reference acid alpha-glucosidase having a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12. 6. The engineered acid alpha-glucosidase of claim 5, wherein the engineered acid alpha- glucosidase exhibits reduced immunogenicity as compared to the reference acid alpha-glucosidase having a sequence corresponding to residues 20 to 944 of SEQ ID NO: 2 or 12, or a sequence corresponding to SEQ ID NO: 2 or 12. 7. The engineered acid alpha-glucosidase of claim 6, wherein the engineered acid alpha- glucosidase exhibits (a) a reduction in Total Immunogenic Score (TIS) of greater than 10 as compared to the reference acid alpha-glucosidase of SEQ ID NO: 2; (b) a reduction in Immunogenic Hit Count (IHC) of greater than 2 as compared to the reference acid alpha-glucosidase of SEQ ID NO: 2; (c) a reduction in Total Immunogenic Score (TIS) of greater than 10 as compared to the reference acid alpha-glucosidase of SEQ ID NO: 12; and/or (d) a reduction in Immunogenic Hit Count (IHC) of greater than 2 as compared to the reference acid alpha-glucosidase of SEQ ID NO: 12. 8. The engineered acid alpha-glucosidase of any one of claims 1-7, comprising a pre- pro-peptide of the engineered acid alpha-glucosidase. Docket No. CX7-223WO1 9. The engineered acid alpha-glucosidase of a claim 8, wherein the pre-pro-peptide of the engineered acid alpha-glucosidase comprises a eukaryotic or synthetic signal peptide sequence. 10. The engineered acid alpha-glucosidase of claim 9, wherein the signal peptide comprises a mouse or human signal peptide sequence. 11. The engineered acid alpha-glucosidase of any one of claims 1-7, comprising a pro- peptide of the engineered acid alpha-glucosidase. 12. The engineered acid alpha-glucosidase of any one of claims 1-11, wherein the engineered acid alpha-glucosidase is purified. 13. A pharmaceutical composition comprising an engineered acid alpha-glucosidase of any one of claims 1-12. 14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable carrier and/or excipient. 15. The pharmaceutical composition of any one of claims 13 or 14, wherein the composition is suitable for parenteral injection or infusion to a human. 16. A recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered acid alpha-glucosidase of any one of claims 1-11. 17. The recombinant polynucleotide of claim 16, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 58-2832 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-753, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-753, wherein the polynucleotide encodes an acid alpha-glucosidase. 18. The recombinant polynucleotide of claim 16 or 17, comprising a polynucleotide sequence codon-optimized for expression of the encoded engineered acid alpha-glucosidase. 19. The recombinant polynucleotide of claim 16, comprising a polynucleotide sequence comprising nucleotide residues 58-2832 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-753, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-753. 20. The recombinant polynucleotide of claim 16, comprising a polynucleotide sequence comprising nucleotide residues 58-2832 of SEQ ID NO: 13, 113, 125, 169, 249, 251, 393, 471, 487, or 505, or a polynucleotide sequence comprising SEQ ID NO: 13, 113, 125, 169, 249, 251, 393, 471, Docket No. CX7-223WO1 487, or 505. 21. An expression vector comprising the recombinant polynucleotide of any one of claims 16-20. 22. The expression vector of claim 21, wherein the recombinant polynucleotide is operably linked to a control sequence. 23. The expression vector of claim 22, wherein the control sequence comprises a promoter. 24. The expression vector of claim 23, wherein the promoter is a heterologous promoter. 25. A host cell comprising the expression vector of any one of claims 21-24. 26. The host cell of claim 25, wherein the host cell is a eukaryotic cell or prokaryotic cell. 27. The host cell of claim 25, wherein the host cell is a mammalian cell. 28. The host cell of claim 27, wherein the mammalian cell is a human cell. 29. The host cell of claim 28, wherein the human cell is from a patient having a deficiency in acid alpha-glucosidase activity. 30. A method of producing an engineered acid alpha-glucosidase variant, comprising culturing the host cell of any one of claims 25-29, under conditions such that the acid alpha- glucosidase encoded by the recombinant polynucleotide is produced. 31. The method of claim 30, further comprising the step of recovering the acid alpha- glucosidase. 32. The method of claim 30 or 31, further comprising the step of purifying the acid alpha- glucosidase. 33. A method for treating and/or preventing symptoms of a deficiency in acid alpha- glucosidase in a subject, comprising administering to a subject in need thereof an effective amount of an engineered acid alpha-glucosidase of any one of claims 1-12, or a pharmaceutical composition of any one of claims 13-15. 34. The method of claim 33, wherein the deficiency in acid alpha-glucosidase is Pompe disease. Docket No. CX7-223WO1 35. The method of any one of claims 33 or 34, wherein the subject is an infant or child. 36. The method of any one of claims 33 or 34, wherein the subject is an adult or young adult. 37. Use of an engineered acid alpha-glucosidase of any one of claims 1-12 for the treatment of a deficiency in acid alpha-glucosidase. 38. Use of an engineered acid alpha-glucosidase of any one of claims 1-12 in the preparation of a medicament for treating a subject with a deficiency in acid alpha-glucosidase. 39. The use of claim 37 or 38, wherein the deficiency in acid alpha-glucosidase is Pompe disease.

Description

Docket No. CX7-223WO1 ENGINEERED ACID ALPHA-GLUCOSIDASE VARIANTS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/511,347, filed June 30, 2023, which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM [0002] The Sequence Listing concurrently submitted herewith as a file name CX7-223WO1_ST26.xml, created on June 27, 2024, with a file size of 2,391,488 bytes, is part of the specification and is incorporated by reference herein. TECHNICAL FIELD [0003] The present disclosure relates to engineered acid alpha-glucosidase (GAA) polypeptides, compositions thereof, polynucleotides encoding the engineered acid alpha-glucosidase polypeptides, and uses of the engineered polypeptides for therapeutic and other purposes. BACKGROUND [0004] Pompe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the gene encoding acid alpha-glucosidase (GAA). This genetic defect leads to reduction in or absence of acid alpha-glucosidase in the body tissues. The resulting accumulation of glycogen in the lysosomes results in lysosomal swelling and rupture, which can lead to cell damage, organelle dysfunction, and other cellular defects. There are two primary forms of Pompe disease, including the classical infantile form and late-onset (childhood or adulthood) form, with some patients displaying an intermediate phenotype. Disease severity is related to the amount of enzyme activity present in the cells of affected individuals. The infantile form is the most severe and a rapidly progressive form, typically with acid alpha-glucosidase activity that is less than 1%, resulting in marked accumulation of glycogen in skeletal muscle as well as heart and other tissues (see, e.g., Hahn and Schänzer, Ann. Transl. Med., 2019, 7:283). In these patients, there is multi-system storage of accumulated lysosomal and non-lysosomal bound- glycogen in the heart, skeletal muscle, and brain tissue (see, e.g., Schoser, Ann. Transl. Med., 2019, 7:292). Patients have elevated creatine kinase levels, hypertrophic cardiomyopathy, failure to thrive, muscular hypotonia, and axial muscle weakness. If untreated, patients typically die within the first year of life due to cardiorespiratory insufficiency. Survival beyond 18 months of age is exceptional. [0005] The infantile form is distinguished from non-classic or late-infantile Pompe disease in which patients display much less severe cardiac hypertrophy. Patients with late-onset Pompe disease typically experience progressive limb-girdle myopathy and respiratory dysfunction. These patients present with predominant, but not exclusive, muscle involvement. The patients eventually become wheelchair and/or ventilator-dependent. Respiratory insufficiency is the leading cause of death in these patients. Some Docket No. CX7-223WO1 patients may synthesize a non-functional form of acid alpha-glucosidase, but others do not produce any cross-reactive immunologic material of the native enzyme. [0006] The human gene encoding acid alpha-glucosidase has been localized to chromosome 17q25.2- q25.3 and has been cloned and sequenced (see, e.g., Peruzzo et al., Ann. Transl. Med., 2019, 7:278-287; and Martiniuk et al., DNA Cell. Biol., 1991, 10:283-292). Although numerous mutations in the gene have been reported, the pathological mechanisms that lead to the wide range of phenotypes observed in affected patients remain unknown. Despite the availability of enzyme replacement therapy (ERT) utilizing recombinant human acid alpha-glucosidase, there remains the need for better treatment and management options for affected patients. SUMMARY [0007] The present disclosure provides acid alpha-glucosidase polypeptides engineered to have improved properties, particularly as compared to the naturally occurring human acid alpha-glucosidase. [0008] In some embodiments, the present disclosure provides an engineered acid alpha-glucosidase polypeptide, or biologically active fragment thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence corresponding to residues 20-944 of an even numbered SEQ ID NO. of SEQ ID NOs: 2, 12, and 14-754, or to an even numbered SEQ ID NO. of SEQ ID NOs: 2, 12, and 14-754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 20-944 of SEQ ID NO: 12 or 2, or relative to the reference sequence corresponding to SEQ ID NO: 12 or 2. [0009] In some embodiments, the engineered acid alpha-glucosidase, or biologically active fragment thereof, comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference sequence correspondin