Chimeric alkaline phosphatase-like proteins
Inventors
Raaben, Willem • Jonk, Luigi Johannes Cornelius • Van Den Berg, Erik Jan • Van Elsas, Andrea • MILLÁN, José Luis
Assignees
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Publication Number
US-11746340-B2
Publication Date
2023-09-05
Expiration Date
Abstract
The invention relates to improved alkaline phosphatases, pharmaceutical compositions comprising improved alkaline phosphatases and the use of improved alkaline phosphatases for preventing, treating or curing diseases.
Core Innovation
The invention relates to a method for improving, reducing or removing symptoms in a subject suffering from a disease accompanied by a local or systemic zinc deficiency by administering a protein having phosphatase activity. The protein is defined by amino acid sequence similarity to multiple reference sequences, including an amino acid sequence at least 200 consecutive amino acids with at least 90% sequence identity with SEQ ID NO: 5, an amino acid sequence at least 50 consecutive amino acids with at least 90% sequence identity with SEQ ID NO: 6, and an amino acid sequence at least 40 consecutive amino acids with at least 90% sequence identity with SEQ ID NO: 7. The full length protein has at least 90% sequence identity with the full length amino acid sequence of SEQ ID NO: 1.
A proviso fixes specific amino acids in the administered protein: the amino acid at position 279 is leucine (L), the amino acid at position 328 is valine (V), and the amino acid at position 478 is leucine (L). The engineered chimeric alkaline phosphatase-like proteins (LVL-RecAP/RecAP) are described with defined sequence identity to SEQ ID NOs and specific residue substitutions, including position 279=L, 328=V, and 478=L. The engineered proteins are presented as having improved zinc-independence at low Zn2+ and enhanced stability/activity in the presence of EDTA.
The background rationale links zinc deficiency to inflammatory and kidney diseases, and the therapeutic scope is defined as diseases selected from sepsis-associated acute kidney injury, ischemic reperfusion kidney damage, or a hypophosphatasia. The document reports that the engineered proteins show superior retained activity versus a reference AP construct under EDTA/chelators, improved kinetics including lower Km for PLP at pH 7.4, increased heat resistance, and favorable organ biodistribution compared with catALPI/crownALPP. The document further reports supportive in vivo efficacy in models including AKI, renal ischemia/reperfusion, and hypophosphatasia in Alp1−/− mice, with outcomes including increased survival, improved body weight, skeletal and dental/corticobone mineralization, and reduced plasma PPi.
Claims Coverage
The independent claim recites a method for improving, reducing or removing symptoms in a subject with a zinc-deficiency–accompanied disease by administering a phosphatase-activity protein defined by multiple conserved sequence regions and specific residue substitutions, with coverage restricted to selected disease categories. The inventive features are anchored by the multi-part sequence identity requirements and the fixed residues at positions 279, 328, and 478.
Multi-region conserved sequence phosphatase protein for zinc-deficiency diseases
Administering a protein having phosphatase activity, where the protein comprises an amino acid sequence of at least 200 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 5, an amino acid sequence of at least 50 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 6, and an amino acid sequence of at least 40 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 7, and where the full length protein comprises an amino acid sequence having at least 90% sequence identity with the full length amino acid sequence of SEQ ID NO: 1.
Fixed residue substitutions at positions 279, 328 and 478
A proviso that the amino acid at position 279 is leucine (L), the amino acid at position 328 is valine (V) and the amino acid at position 478 is leucine (L).
Treatment of selected zinc-deficiency–accompanied diseases
Improving, reducing or removing symptoms in a subject suffering from a disease accompanied by a local or systemic zinc deficiency, wherein said disease is selected from sepsis-associated acute kidney injury, ischemic reperfusion kidney damage or a hypophosphatasia.
Across the dependent claims described in the provided content, coverage is refined by increasing sequence identity thresholds and narrowing consecutive amino-acid segment lengths while maintaining the fixed residues at positions 279, 328 and 478, and by restricting the hypophosphatasia category or requiring use of the amino acid sequence of SEQ ID NO: 1.
Stated Advantages
Improved zinc-independence at low Zn2+ and enhanced stability/activity in the presence of EDTA.
Superior retained phosphatase activity versus a reference AP construct under EDTA/chelators.
Improved kinetics, including lower Km for PLP at pH 7.4.
Increased heat resistance.
Favorable organ biodistribution compared with catALPI/crownALPP.
Demonstrated in vivo efficacy in models including AKI, renal ischemia/reperfusion, and hypophosphatasia, including outcomes such as increased survival and improved mineralization.
Documented Applications
A method for improving, reducing or removing symptoms in sepsis-associated acute kidney injury and ischemic reperfusion kidney damage in subjects with local or systemic zinc deficiency.
A method for treating hypophosphatasia in subjects with local or systemic zinc deficiency, including forms selected from perinatal, infantile, childhood, or adult hypophosphatasia.
In vivo use supported by models including LPS-induced AKI (rats), renal ischemia/reperfusion kidney damage (pig model endpoints), and hypophosphatasia in Alp1−/− mice.
Support by a described pharmacokinetic/safety study setup in healthy humans for LVL-recAP.
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