Human enzyme-mediated homocysteine depletion for the treatment of patients with hyperhomocysteinemia and homocystinuria
阅读说明：本技术 用于治疗高同型半胱氨酸血症和同型胱胺酸尿症患者的人类酶介导的同型半胱氨酸耗竭 (Human enzyme-mediated homocysteine depletion for the treatment of patients with hyperhomocysteinemia and homocystinuria ) 是由 G·乔治乌 E·斯通 W-C·路 于 2018-05-11 设计创作，主要内容包括：描述了与具有同型(半)胱氨酸酶活性的改良蛋白的工程化有关的方法和组合物。例如,公开了包含一个或多个氨基酸取代并且能够降解同型(半)胱氨酸的经修饰的胱硫醚-γ-裂解酶(CGL)。此外,提供了使用所公开的酶或核酸,利用同型(半)胱氨酸耗竭来治疗同型胱胺酸尿症或高同型半胱氨酸血症的组合物和方法。(Methods and compositions related to the engineering of improved proteins having homotypic (cysteine) enzyme activity are described. For example, modified cystathionine-gamma-lyases (CGLs) comprising one or more amino acid substitutions and capable of degrading homotypic cystine(s) are disclosed. In addition, compositions and methods for treating homocysteinuria or hyperhomocysteinemia using homotypic (cysteine) depletion using the disclosed enzymes or nucleic acids are provided.)
1. An isolated modified primate cystathionine- γ -lyase (CGL) comprising at least the following substitutions relative to a native human CGL amino acid sequence (SEQ ID NO:1), wherein the modified enzyme has both homocysteinase activity and homocysteinase activity, said substitutions being selected from the group consisting of:
(a) isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353;
(b) asparagine at position 59, leucine at position 119, aspartic acid at position 336, and valine at position 339;
(c) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(d) isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(e) asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(f) isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(g) asparagine at position 59, leucine at position 119, glutamic acid at position 336, and valine at position 339;
(h) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(i) isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(j) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(i) asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(k) isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(l) Isoleucine at position 59, leucine at position 63, methionine at position 91, histidine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353; and
(m) isoleucine at position 59, leucine at position 63, methionine at position 91, glycine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
2. The enzyme of claim 1, wherein the substitutions comprise asparagine at position 59, leucine at position 119, aspartic acid at position 336, and valine at position 339.
3. The enzyme of claim 2, wherein the substitutions further comprise leucine at position 63, methionine at position 91, arginine at position 268, glycine at position 311, and serine at position 353.
4. The enzyme of claim 1, wherein the substitutions comprise asparagine at position 59, leucine at position 119, glutamic acid at position 336, and valine at position 339.
5. The enzyme of claim 4, wherein the substitutions further comprise leucine at position 63, methionine at position 91, arginine at position 268, glycine at position 311, and serine at position 353.
6. The enzyme of claim 1, wherein the substitution comprises isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
7. The enzyme of claim 1, wherein the substitution comprises isoleucine at position 59, leucine at position 63, methionine at position 91, histidine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
8. The enzyme of claim 1, wherein the substitution comprises isoleucine at position 59, leucine at position 63, methionine at position 91, glycine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
9. The enzyme of claim 1, wherein the modified CGL enzyme is a modified sumanshan chimpanzee CGL enzyme.
10. The enzyme of claim 9, wherein the modified sumatriptan CGL enzyme comprises a substitution selected from the group consisting of:
(a) V59I, S63L, L91M, R119D, K268R, T311G, E339V and V353S;
(b) V59N, R119L, T336D and E339V;
(c) V59N, S63L, L91M, R119L, K268R, T311G, T336D, E339V and V353S;
(d) V59I, S63L, L91M, R119L, K268R, T311G, T336D, E339V and V353S;
(e) V59N, S63L, L91M, R119A, K268R, T311G, T336D, E339V and V353S;
(f) V59I, S63L, L91M, R119A, K268R, T311G, T336D, E339V and V353S;
(g) V59N, R119L, T336E and E339V;
(h) V59N, S63L, L91M, R119L, K268R, T311G, T336E, E339V and V353S;
(i) V59I, S63L, L91M, R119L, K268R, T311G, T336E, E339V and V353S;
(j) V59N, S63L, L91M, R119A, K268R, T311G, T336E, E339V and V353S;
(k) V59I, S63L, L91M, R119A, K268R, T311G, T336E, E339V and V353S;
(l) V59I, S63L, L91M, R119H, K268R, T311G, E339V and V353S; and
(m) V59I, S63L, L91M, R119G, K268R, T311G, E339V and V353S.
11. The enzyme of claim 1, wherein the modified CGL enzyme is a modified human CGL enzyme, a modified cynomolgus monkey CGL enzyme, a modified chimpanzee CGL enzyme, or a modified bonobo CGL enzyme.
12. The enzyme of claim 11, wherein the modified human CGL enzyme, modified cynomolgus CGL enzyme, modified chimpanzee CGL enzyme, or modified bonobo CGL enzyme comprises a substitution selected from the group consisting of:
(a) E59I, S63L, L91M, R119D, K268R, T311G, E339V, and I353S;
(b) E59N, R119L, T336D and E339V;
(c) E59N, S63L, L91M, R119L, K268R, T311G, T336D, E339V, and I353S;
(d) E59I, S63L, L91M, R119L, K268R, T311G, T336D, E339V, and I353S;
(e) E59N, S63L, L91M, R119A, K268R, T311G, T336D, E339V, and I353S;
(f) E59I, S63L, L91M, R119A, K268R, T311G, T336D, E339V, and I353S;
(g) E59N, R119L, T336E and E339V;
(h) E59N, S63L, L91M, R119L, K268R, T311G, T336E, E339V, and I353S;
(i) E59I, S63L, L91M, R119L, K268R, T311G, T336E, E339V, and I353S;
(j) E59N, S63L, L91M, R119A, K268R, T311G, T336E, E339V, and I353S;
(k) E59I, S63L, L91M, R119A, K268R, T311G, T336E, E339V, and I353S;
(l) E59I, S63L, L91M, R119H, K268R, T311G, E339V, and I353S; and
(m) E59I, S63L, L91M, R119G, K268R, T311G, E339V, and I353S.
13. The enzyme of any one of claims 1 to 8, further comprising a heterologous peptide fragment or polysaccharide.
14. The enzyme of claim 13, wherein the heterologous peptide fragment is an XTEN polypeptide, IgGFc, albumin, or albumin binding peptide.
15. The enzyme of claim 13, wherein the polysaccharide comprises a polysialic acid polymer.
16. The enzyme of any one of claims 1-15, wherein the enzyme is conjugated to polyethylene glycol (PEG).
17. The enzyme of claim 16, wherein the enzyme is coupled to PEG via one or more lysine residues.
18. A nucleic acid comprising a nucleotide sequence encoding the enzyme of any one of claims 1-14.
19. The nucleic acid of claim 18, wherein the nucleic acid is codon optimized for expression in bacteria, fungi, insects, or mammals.
20. The nucleic acid of claim 19, wherein the bacterium is e.
21. The nucleic acid of claim 20, wherein the nucleic acid comprises one of SEQ ID No. 64 to SEQ ID No. 66.
22. An expression vector comprising the nucleic acid of claim 18 or 19.
23. A host cell comprising the nucleic acid of claim 18 or 19.
24. The host cell of claim 23, wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.
25. A therapeutic formulation comprising the enzyme of any one of claims 1-17 or the nucleic acid of any one of claims 18 or 19 in a pharmaceutically acceptable carrier.
26. A method of treating a subject having or at risk of developing homocysteinuria or hyperhomocysteinemia with homocysteinuria or hyperhomocysteinemia comprising administering to the subject a therapeutically effective amount of the formulation of claim 25.
27. A method of treating a subject having or at risk of developing homocysteinuria or hyperhomocysteinemia with homocysteinuria or hyperhomocysteinemia, said method comprising:
administering to the subject a therapeutically effective amount of the formulation of claim 25, or a formulation comprising an isolated, modified primate cystathionine- γ -lyase (CGL) having a substitution relative to the native human CGL amino acid sequence (SEQ id no:1), or a nucleic acid comprising a nucleotide sequence encoding the modified enzyme, the substitution selected from the group consisting of:
(a) asparagine at position 59, leucine at position 119, and valine at position 339;
(b) valine at position 59, leucine at position 119, and valine at position 339;
(c) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353;
(d) isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353;
(e) asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353; and
(f) isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
28. The method of claim 27, wherein the modified CGL enzyme is a modified sumanshan chimpanzee CGL enzyme.
29. The method of claim 28, wherein the modified sumatriptan CGL enzyme comprises a substitution selected from the group consisting of:
(a) V59N, R119L and E339V;
(b) R119L and E339V;
(c) V59N, S63L, L91M, R119L, K268R, T311G, E339V and V353S;
(d) V59I, S63L, L91M, R119L, K268R, T311G, E339V and V353S;
(e) V59N, S63L, L91M, R119A, K268R, T311G, E339V and V353S; and
(f) V59I, S63L, L91M, R119A, K268R, T311G, E339V and V353S.
30. The method of claim 27, wherein the modified CGL enzyme is a modified human CGL enzyme, a modified cynomolgus monkey CGL enzyme, a modified chimpanzee CGL enzyme, or a modified bonobo CGL enzyme.
31. The method of claim 30, wherein the modified human CGL enzyme, modified cynomolgus CGL enzyme, modified chimpanzee CGL enzyme, or modified bonobo CGL enzyme comprises a substitution selected from the group consisting of:
(a) E59N, R119L and E339V;
(b) E59V, R119L and E339V;
(c) E59N, S63L, L91M, R119L, K268R, T311G, E339V, and I353S;
(d) E59I, S63L, L91M, R119L, K268R, T311G, E339V, and I353S;
(e) E59N, S63L, L91M, R119A, K268R, T311G, E339V, and I353S; and
(f) E59I, S63L, L91M, R119A, K268R, T311G, E339V, and I353S.
32. The method of any one of claims 27-31, wherein the enzyme further comprises a heterologous peptide fragment.
33. The method of claim 32, wherein the heterologous peptide fragment is an XTEN polypeptide, an IgG Fc, albumin, an albumin binding peptide, or polysialic acid time extension.
34. The method of any one of claims 27-31, wherein the enzyme is conjugated to polyethylene glycol (PEG).
35. The method of claim 34, wherein the enzyme is coupled to PEG through one or more lysine or cysteine residues.
36. The method of claim 27, wherein the subject is maintained on a methionine restricted diet.
37. The method of claim 27, wherein the subject maintains a normal diet.
38. The method of claim 27, wherein the subject is a human patient.
39. The method of claim 27, wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intralesionally, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intramuscularly, intravesicularly, intraumbilically, by injection, infusion, continuous infusion, direct local perfusion bathing target cells, or via a catheter.
40. The method of claim 27, wherein the subject has previously been treated for homocystinuria or hyperhomocysteinemia and the enzyme is administered to prevent the recurrence of the homocystinuria or hyperhomocysteinemia.
41. The method of claim 27, further comprising administering to the subject at least a second homocystinuria or hyperhomocysteinemia therapy.
42. The method of claim 41, wherein said second homocystinuria or hyperhomocysteinemia therapy is high dose vitamin B6 or betaine (N, N, N-trimethylglycine) therapy.
43. The therapeutic preparation according to claim 25 for use as a medicament for treating a subject suffering from homocysteinuria or hyperhomocysteinemia.
44. The enzyme of any one of claims 1-14 for use in treating homocysteinuria or hyperhomocysteinemia in a subject.
Technical Field
Recombinantly engineered primate enzyme variants with homocysteine degrading activity and stability suitable for human therapy are disclosed. Also provided are compositions and methods for treating homocysteinuria and hyperhomocysteinemia with enzymes that degrade homocysteine and homocysteine.
Description of the related Art
Homocysteinuria is a rare disease caused by a congenital metabolic defect involving sulfur amino acids. Typical homocystinuria, the most common form, is caused by a deficiency in cystathionine beta-synthase (CBS) (Mudd et al, 1964), whereas non-classical homocystinuria is often associated with a deficiency in various enzymes involved in folate metabolism (e.g., MTHFR, MTRR or MTR) (Kang et al, 1987). Patients with homocysteine urine often exhibit thromboembolism, cognitive disorders, osteoporosis, and lens dislocation (Kruger et al, 2003). Patients also suffer from hyperhomocysteinemia, a condition in which serum homocysteine (Hcy) concentrations exceed 15 μ M. Patients with typical homocysteinuria and some atypical forms often exhibit elevated serum methionine (Met) levels, while cystathionine (Cth) and cysteine (Cys) levels are low (Kruger et al, 2003). It is estimated that one in every 344,000 people worldwide is affected, but in some countries its incidence is much higher. The most common medical condition associated with typical homocysteinuria is cardiovascular complications, including increased risk of blood clots. Other symptoms include skeletal deformities, intraocular lens dislocation, and developmental and learning deficiencies.
Currently available methods for treating typical homocysteinuria include methionine restricted diets as well as high doses of vitamin B6 and betaine (N, N-trimethylglycine), the latter reducing homocysteine levels. These treatments focus on preventing the accumulation of Hcy and show moderate to limited efficacy (Walter et al, 1998). Although these therapies may be effective in some patients, the response fluctuates significantly due to the changes in the genetic mutations that drive the disease. As a result, there is an opportunity to develop a therapy that will meet the needs of all patients by lowering homocysteine levels in the blood back to the normal range. Thus, there is a need for new methods and compositions for treating these patients.
Disclosure of Invention
Methods are disclosed for the efficient degradation of homo (cysteine) using engineered human cystathionine-gamma-lyase (CGL) to allow for appropriate therapy for patients with homocysteinuria and hyperhomocysteinemia by degrading excess serum homo (cysteine) and providing a sink of intracellular homo (cysteine). The method can reduce serum homocysteine level (tHcy) to a level of less than 25. mu.M.
Methods of treatment, particularly treatment of subjects with homocysteinuria or hyperhomocysteinemia, are provided that include administering a modified CGL enzyme, a nucleic acid encoding a modified CGL enzyme, or a formulation comprising a modified CGL enzyme in a gene therapy vector. The subject can be any animal, such as a mouse. For example, the subject can be a mammal, rodent, primate, or human patient. The method may comprise selecting a patient having homocysteinuria or hyperhomocysteinemia. The subject or patient may be maintained on a methionine restricted diet or on a normal diet in conjunction with treatment with the composition.
Provided herein are human cystathionine-gamma-lyase (hCGL) mutants having catalytic activity for homotypic cystine for use in therapeutic methods. For example, an enzyme variant may have an amino acid sequence selected from the group consisting of: 2-6 and 37-39 of SEQ ID NO. In particular, the variant may be derived from a human enzyme, such as human cystathionine- γ -lyase (CGL). Polypeptides comprising modified human CGLs capable of degrading homotypic (cysteine) are provided. The polypeptide may be capable of degrading homotypic (cysteine) under physiological conditions. For example, the polypeptide may exhibit catalytic activity towards L-homocystine up to kcat/KMTo achieve 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8,7, 6, 5,4, 3, 2,1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001mM-1s-1Or any range derived therein.
These enzymes are engineered by introducing amino acid substitutions in the human enzyme cystathionine-gamma-lyase (CGL). Variants with amino acid substitutions include SEQ ID NO 2, hCGL-E59N-R119L-E339V (hCGL-NLV); 3, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8 mut-1); 4, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8 mut-2); 5, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8 mut-3); 6, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8 mut-4); 27, hCGL-E59N-R119L-T336D-E339V (hCGL-NLDV); 28, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-1); 29, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-2); 30, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-3); 31, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-4); 32, hCGL-E59N-R119L-T336E-E339V (hCGL-NLEV); 33, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-1); 34, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-2); 35, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-3); 36, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-4); 37, hCGL-E59I-S63L-L91M-R119D-K268R-T311G-E339V-I353S (mutant 3); 38, hCGL-E59I-S63L-L91M-R119H-K268R-T311G-E339V-I353S (mutant 4); and SEQ ID NO 39, hCGL-E59I-S63L-L91M-R119G-K268R-T311G-E339V-I353S (mutant 5).
The modified CGL enzymes discussed herein can be characterized as having a percentage of identity compared to an unmodified CGL enzyme (e.g., a native CGL enzyme). For example, the unmodified CGL enzyme may be a native primate cystathionase (i.e., cystathionine- γ -lyase). The percentage identity between the unmodified portion of the modified CGLase (i.e., the sequence of the modified CGLase, excluding any substitutions at amino acids at positions 59, 63, 91, 119, 268, 311, 336, 339 and/or 353 of SEQ ID No:1 and the homology thereto of SEQ ID Nos: 7-10, see FIG. 6) and the native CGLase may be at least 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein). It is also contemplated that the percent identity discussed above may relate to a particular modified region of an enzyme as compared to an unmodified region of the corresponding native enzyme. For example, a modified CGL enzyme may comprise a modified or mutated substrate recognition site, which may be characterized based on the identity of the amino acid sequence of the modified or mutated substrate recognition site to the amino acid sequence of an unmodified or native CGL enzyme from the same species or across species. For example, a modified human CGL enzyme characterized as having at least 90% identity to an unmodified human CGL enzyme means that at least 90% of the amino acids in the modified human CGL enzyme are the same as the amino acids in the unmodified human CGL enzyme.
The unmodified CGL enzyme may be a native CGL enzyme, particularly a human isoform or other primate isoforms. For example, a native human CGL enzyme may have SEQ ID NO:1 non-limiting examples of other native primate CGL enzymes include Sumenda chimpanzee CGL (Genbank ID: NP-001124635.1; SEQ ID NO:7), cynomolgus monkey CGL (Genbank ID: AAW 71993.1; SEQ ID NO:8), chimpanzee CGL (Genbank ID: XP-513486.2; SEQ ID NO:9), and bonobo CGL (Genbank ID: XP-003830652.1; SEQ ID NO: 10). Exemplary native CGL enzymes include sequences having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity (or any range derivable therein) to SEQ ID NOs:1 or 7-10.
The native CGL may be modified by one or more other modifications, such as chemical modifications, substitutions, insertions, deletions and/or truncations. The modification may be at the substrate recognition site of the native enzyme. Native CGLs may be modified by substitution. For example, the number of substitutions may be four, five, six, seven or more. The native CGL may be modified at the substrate recognition site or at any position that may affect substrate specificity. The modified polypeptide may have at least one amino acid substitution at an amino acid position corresponding to E59, S63, L91, R119, K268, T311, T336, E339 and/or I353 of SEQ ID No. 1 or 59, 63, 91, 119, 268, 311, 336, 339 and/or 353 of primate CGL. For example, the primate may be human, sumatran, cynomolgus, chimpanzee or bonobo. For example, the equivalent substitution in SEQ ID NO 1 for E59 for SEQ ID NO 7 would modify the amino acid sequence as shown in SEQ ID NO:1 instead of glutamic acid 1. Another expected substitution of SEQ ID NO:7 is V353, which is an isoleucine at the equivalent position of SEQ ID NO:1 as shown in FIG. 6.
Substitutions in the modified CGL enzyme may occur at amino acids 59, 63, 91, 119, 268, 311, 339 and/or 353, and may be aspartic acid (N), valine (V), leucine (L), methionine (M), arginine (R), glycine (G), alanine (a) or serine (S). The modification may be selected from the group consisting of: one or more substitutions of E/V59N, E59I, S63L, L91M, R119L, R119A, R119D, R119H, R119G, K268R, T311G, T336D, T336E, E339V, and I/V353S. Substitutions may include S63L, L91M, K268R, T311G, E339V and I/V353S substitutions. The substitutions may comprise further substitutions of E/V59N or E/V59I; any one of R119L, R119A, R119D, R119H, and R119G; and/or T336D or T336E.
The substituent may be a combination of E59, S63, L91, R119, K268, T311, I353 and E339 of human CGL (e.g., a modified polypeptide having the amino acid sequence of SEQ ID NO:3, a fragment or homolog thereof), a combination of E59, S63, L91, R119, K268, T311, E339 and I353 of human CGL (e.g., a modified polypeptide having the amino acid sequence of SEQ ID NO:4, a fragment or homolog thereof), a combination of E59, S63, L91, R119, K268, T311, E339 and I353 of human CGL (e.g., a modified polypeptide having the amino acid sequence of SEQ ID NO:5, a fragment or homolog thereof), a combination of E59, S63, L91, R119, K268, T311, E339 and I353 of human CGL (e.g., a modified polypeptide having the amino acid sequence of SEQ ID NO:6, a fragment or homolog thereof), a combination of E59, S63, S119, R119, K268 and I353 (e.g., a combination of E59, S63, R119, K268, T311, T353 and I353 of human CGL 1, has the sequence shown in SEQ ID NO:37, a fragment thereof, or a homolog thereof), a combination of E59I, S63L, L91M, R119H, K268R, T311G, E339V, and I353S (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:38, a fragment thereof, or a homolog thereof), a combination of E59I, S63L, L91M, R119G, K268R, T311G, E339V, and I353S (e.g., a polypeptide having the amino acid sequence of SEQ ID NO:39, a fragment thereof or a homologue thereof), or the amino acid sequence of SEQ ID NO:2-6 and 37-39 bind to either of these modifications of T336D or T336E. The modified CGL enzyme may be a Sumendong chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO: 11), a Sumendong chimpanzee CGL-ILMLRGVS mutant (SEQ ID NO: 12), a Sumendong chimpanzee CGL-NLMARGVS mutant (SEQ ID NO:13), a Sumendong chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:14), a Sumendong chimpanzee CGL-MARGVS mutant (SEQ ID NO:40), a Sumendong chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:41), a Sumendong chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:42), a Sumendong chimpanzee CGL-MARNLGVS mutant (SEQ ID NO:52), a Sumendong chimpanzee CGL-NLMARGVS mutant (SEQ ID NO:53), a Sumendong chimpanzee CGL-NLGVS mutant (SEQ ID NO:54), a chimpanzee CGL-NLGVS mutant (SEQ ID NO: 15), Macaca fascicularis CGL-ILMARGVS mutant (SEQ ID NO:16), Macaca fascicularis CGL-NLMLRGVS mutant (SEQ ID NO:17), Macaca fascicularis CGL-ILMARGVS mutant (SEQ ID NO:18), Macaca fascicularis CGL-ILMARGVS mutant (SEQ ID NO:43), Macaca fascicularis CGL-ILMARGVS mutant (SEQ ID NO:44), Macaca fascicularis CGL-ILMARGVS mutant (SEQ ID NO:45), Macaca fascicularis CGL-NLRGVS mutant (SEQ ID NO:55), Macaca fascicularis CGL-NLRGVS mutant (SEQ ID NO:56), Macaca fascicularis CGL-NLRGVS mutant (SEQ ID NO:57), chimpanzee CGL-NLRGVS mutant (SEQ ID NO:19), chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:20), chimpanzee CGL-NLGVS mutant (SEQ ID NO:21), chimpanzee CGL-NLGVS mutant (SEQ ID NO:22), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:46), chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:47), chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:48), chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:58), chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:59), chimpanzee CGL-NLRGVS mutant (SEQ ID NO:60), bonobo CGL-NLRGVS mutant (SEQ ID NO:23), bonobo CGL-ILMARS mutant (SEQ ID NO:24), bonobo CGL-NLRGVS mutant (SEQ ID NO:25), bonobo CGL-ILGVS mutant (SEQ ID NO:26), bonobo CGL-ILMARGVS mutant (SEQ ID NO:49), bonobo CGL-ILMARGVS mutant (SEQ ID NO:51), or bonobo CGL-NLRGVS mutant (SEQ ID NO:51), Bonobo CGL-NLMDRGVS mutant (SEQ ID NO:61), bonobo CGL-NLMDRGVS mutant (SEQ ID NO:62), and bonobo CGL-NLMDRGVS mutant (SEQ ID NO: 63).
The modified CGL enzyme may be linked to a heterologous amino acid sequence. For example, the modified CGL enzyme may be linked to a heterologous amino acid sequence as a fusion protein. The modified CGL enzyme may be linked to an amino acid sequence, such as IgG Fc, albumin binding peptide, polysialic acid time extension, or XTEN polypeptide, to increase half-life in vivo.
To increase serum stability, the modified CGL enzyme may be linked to one or more polyether molecules. The polyether may be polyethylene glycol (PEG). The modified CGL enzyme may be attached to PEG through specific amino acid residues such as lysine or cysteine. For therapeutic administration, such a polypeptide comprising a modified CGL enzyme may be dispersed in a pharmaceutically acceptable carrier.
Nucleic acids encoding modified CGL enzymes are contemplated. The nucleic acid may be codon optimized for expression in bacteria, for example in e. Nucleic acids codon-optimized for expression of the modified CGL enzymes provided in SEQ ID NOS 37-39 in E.coli are provided in SEQ ID NOS 64-66, respectively. The sequence provided in SED ID NO 64-66 encodes an N-terminal 6 x-histidine tag. Thus, the sequence represented by SEQ ID NO: the twelfth and following amino acids encoded by 64-66 correspond to SEQ ID nos: 37-39 and the following amino acids. Alternatively, the nucleic acid can be codon optimized for expression in fungi (e.g., yeast), insects, or mammals. Vectors, such as expression vectors, comprising such nucleic acids are also contemplated. The nucleic acid encoding the modified CGL enzyme may be operably linked to a promoter, including but not limited to a heterologous promoter. The modified CGL enzyme may be delivered to a target cell via a vector (e.g., a gene therapy vector). Such vectors may be modified by recombinant DNA techniques to achieve expression of the modified CGL-encoding nucleic acid in a target cell. These vectors may be derived from vectors of non-viral (e.g., plasmid) or viral (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes virus, or vaccinia virus) origin. Non-viral vectors may be complexed with agents to facilitate DNA entry across cell membranes. Examples of such non-viral vector complexes include formulations with polycationic agents that facilitate condensation of DNA, as well as lipid-based delivery systems. Lipid-based delivery systems include the delivery of liposome-based nucleic acids.
Host cells comprising such vectors are provided. The host cell can be a bacterium (e.g., E.coli), a fungal cell (e.g., yeast), an insect cell, or a mammalian cell.
The vector may be introduced into a host cell to express the modified CGL enzyme. The protein may be expressed in any suitable manner. The protein may be expressed in a host cell such that the protein is glycosylated. Alternatively, the protein may be expressed in a host cell such that the protein is unglycosylated.
The therapeutic formulations containing the modified CGL enzyme may be administered intravenously, intradermally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, by infusion, continuous infusion, via a catheter in a lipid composition (e.g., liposomes). The subject may have previously been treated for homocysteinuria or hyperhomocysteinemia. Administering the enzyme to treat or ameliorate the recurrence of homocysteinuria or hyperhomocysteinemia; administration of a compound or composition described herein may ameliorate one or more conditions and/or symptoms associated with homocysteinuria and hyperhomocysteinemia. Known symptoms of homocysteinuria include intraocular lens dislocation, myopia, abnormal blood clots, osteoporosis, learning disorders, developmental problems, chest deformities, scoliosis, megaloblastic anemia, and seizures. Known symptoms of hyperhomocysteinemia include blood clots, impaired vascular lining, dementia (e.g., alzheimer's disease), and bone fractures.
The method may further comprise administering at least a second therapy to the subject, for example a second homocystinuria or hyperhomocysteinemia therapy. The second therapy for homocysteinuria or hyperhomocysteinemia may be a high dose vitamin B6 or betaine (N, N-trimethylglycine) therapy.
Compositions comprising a modified CGL enzyme or a nucleic acid encoding a modified CGL enzyme are provided for treating homocysteinuria or hyperhomocysteinemia in a subject. Provides the use of a modified CGL enzyme or a nucleic acid encoding a modified CGL enzyme in the manufacture of a medicament for the treatment of homocysteinuria or hyperhomocysteinemia. The modified CGL enzyme may be any modified CGL enzyme disclosed herein.
Also provided herein are:
1. an isolated modified primate cystathionine- γ -lyase (CGL) comprising at least the following substitutions relative to a native human CGL amino acid sequence (SEQ ID NO:1), wherein the modified enzyme has both homocysteinase activity and homocysteinase activity, said substitutions being selected from the group consisting of:
(a) isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353;
(b) asparagine at position 59, leucine at position 119, aspartic acid at position 336, and valine at position 339;
(c) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(d) isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(e) asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(f) isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353;
(g) asparagine at position 59, leucine at position 119, glutamic acid at position 336, and valine at position 339;
(h) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(i) isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(j) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(i) asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(k) isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353;
(l) Isoleucine at position 59, leucine at position 63, methionine at position 91, histidine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353; and
(m) isoleucine at position 59, leucine at position 63, methionine at position 91, glycine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
2. The enzyme of claim 1, wherein the substitutions comprise asparagine at position 59, leucine at position 119, aspartic acid at position 336, and valine at position 339.
3. The enzyme of claim 2, wherein the substitutions further comprise leucine at position 63, methionine at position 91, arginine at position 268, glycine at position 311, and serine at position 353.
4. The enzyme of claim 1, wherein the substitutions comprise asparagine at position 59, leucine at position 119, glutamic acid at position 336, and valine at position 339.
5. The enzyme of claim 4, wherein the substitutions further comprise leucine at position 63, methionine at position 91, arginine at position 268, glycine at position 311, and serine at position 353.
6. The enzyme of claim 1, wherein the substitution comprises isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
7. The enzyme of claim 1, wherein the substitution comprises isoleucine at position 59, leucine at position 63, methionine at position 91, histidine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
8. The enzyme of claim 1, wherein the substitution comprises isoleucine at position 59, leucine at position 63, methionine at position 91, glycine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
9. The enzyme of claim 1, wherein the modified CGL enzyme is a modified sumanshan chimpanzee CGL enzyme.
10. The enzyme of claim 9, wherein the modified sumatriptan CGL enzyme comprises a substitution selected from the group consisting of:
(a) V59I, S63L, L91M, R119D, K268R, T311G, E339V and V353S;
(b) V59N, R119L, T336D and E339V;
(c) V59N, S63L, L91M, R119L, K268R, T311G, T336D, E339V and V353S;
(d) V59I, S63L, L91M, R119L, K268R, T311G, T336D, E339V and V353S;
(e) V59N, S63L, L91M, R119A, K268R, T311G, T336D, E339V and V353S;
(f) V59I, S63L, L91M, R119A, K268R, T311G, T336D, E339V and V353S;
(g) V59N, R119L, T336E and E339V;
(h) V59N, S63L, L91M, R119L, K268R, T311G, T336E, E339V and V353S;
(i) V59I, S63L, L91M, R119L, K268R, T311G, T336E, E339V and V353S;
(j) V59N, S63L, L91M, R119A, K268R, T311G, T336E, E339V and V353S;
(k) V59I, S63L, L91M, R119A, K268R, T311G, T336E, E339V and V353S;
(l) V59I, S63L, L91M, R119H, K268R, T311G, E339V and V353S; and
(m) V59I, S63L, L91M, R119G, K268R, T311G, E339V and V353S.
11. The enzyme of claim 1, wherein the modified CGL enzyme is a modified human CGL enzyme, a modified cynomolgus monkey CGL enzyme, a modified chimpanzee CGL enzyme, or a modified bonobo CGL enzyme.
12. The enzyme of claim 11, wherein the modified human CGL enzyme, modified cynomolgus CGL enzyme, modified chimpanzee CGL enzyme, or modified bonobo CGL enzyme comprises a substitution selected from the group consisting of:
(a) E59I, S63L, L91M, R119D, K268R, T311G, E339V, and I353S;
(b) E59N, R119L, T336D and E339V;
(c) E59N, S63L, L91M, R119L, K268R, T311G, T336D, E339V, and I353S;
(d) E59I, S63L, L91M, R119L, K268R, T311G, T336D, E339V, and I353S;
(e) E59N, S63L, L91M, R119A, K268R, T311G, T336D, E339V, and I353S;
(f) E59I, S63L, L91M, R119A, K268R, T311G, T336D, E339V, and I353S;
(g) E59N, R119L, T336E and E339V;
(h) E59N, S63L, L91M, R119L, K268R, T311G, T336E, E339V, and I353S;
(i) E59I, S63L, L91M, R119L, K268R, T311G, T336E, E339V, and I353S;
(j) E59N, S63L, L91M, R119A, K268R, T311G, T336E, E339V, and I353S;
(k) E59I, S63L, L91M, R119A, K268R, T311G, T336E, E339V, and I353S;
(l) E59I, S63L, L91M, R119H, K268R, T311G, E339V, and I353S; and
(m) E59I, S63L, L91M, R119G, K268R, T311G, E339V, and I353S.
13. The enzyme of any one of claims 1 to 8, further comprising a heterologous peptide fragment or polysaccharide.
14. The enzyme of claim 13, wherein the heterologous peptide fragment is an XTEN polypeptide, IgGFc, albumin, or albumin binding peptide.
15. The enzyme of claim 13, wherein the polysaccharide comprises a polysialic acid polymer.
16. The enzyme of any one of claims 1-15, wherein the enzyme is conjugated to polyethylene glycol (PEG).
17. The enzyme of claim 16, wherein the enzyme is coupled to PEG via one or more lysine residues.
18. A nucleic acid comprising a nucleotide sequence encoding the enzyme of any one of claims 1-14.
19. The nucleic acid of claim 18, wherein the nucleic acid is codon optimized for expression in bacteria, fungi, insects, or mammals.
20. The nucleic acid of claim 19, wherein the bacterium is e.
21. The nucleic acid of claim 20, wherein the nucleic acid comprises one of SEQ ID No. 64 to SEQ ID No. 66.
22. An expression vector comprising the nucleic acid of claim 18 or 19.
23. A host cell comprising the nucleic acid of claim 18 or 19.
24. The host cell of claim 23, wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.
25. A therapeutic formulation comprising the enzyme of any one of claims 1-17 or the nucleic acid of any one of claims 18 or 19 in a pharmaceutically acceptable carrier.
26. A method of treating a subject having or at risk of developing homocysteinuria or hyperhomocysteinemia with homocysteinuria or hyperhomocysteinemia comprising administering to the subject a therapeutically effective amount of the formulation of claim 25.
27. A method of treating a subject having or at risk of developing homocysteinuria or hyperhomocysteinemia with homocysteinuria or hyperhomocysteinemia, said method comprising:
administering to the subject a therapeutically effective amount of the formulation of claim 25, or a formulation comprising an isolated, modified primate cystathionine- γ -lyase (CGL) having a substitution relative to the native human CGL amino acid sequence (SEQ ID NO:1), or a nucleic acid comprising a nucleotide sequence encoding the modified enzyme, the substitution selected from the group consisting of:
(a) asparagine at position 59, leucine at position 119, and valine at position 339;
(b) valine at position 59, leucine at position 119, and valine at position 339;
(c) asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353;
(d) isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353;
(e) asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353; and
(f) isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.
28. The method of claim 27, wherein the modified CGL enzyme is a modified sumanshan chimpanzee CGL enzyme.
29. The method of claim 28, wherein the modified sumatriptan CGL enzyme comprises a substitution selected from the group consisting of:
(a) V59N, R119L and E339V;
(b) R119L and E339V;
(c) V59N, S63L, L91M, R119L, K268R, T311G, E339V and V353S;
(d) V59I, S63L, L91M, R119L, K268R, T311G, E339V and V353S;
(e) V59N, S63L, L91M, R119A, K268R, T311G, E339V and V353S; and
(f) V59I, S63L, L91M, R119A, K268R, T311G, E339V and V353S.
30. The method of claim 27, wherein the modified CGL enzyme is a modified human CGL enzyme, a modified cynomolgus monkey CGL enzyme, a modified chimpanzee CGL enzyme, or a modified bonobo CGL enzyme.
31. The method of claim 30, wherein the modified human CGL enzyme, modified cynomolgus CGL enzyme, modified chimpanzee CGL enzyme, or modified bonobo CGL enzyme comprises a substitution selected from the group consisting of:
(a) E59N, R119L and E339V;
(b) E59V, R119L and E339V;
(c) E59N, S63L, L91M, R119L, K268R, T311G, E339V, and I353S;
(d) E59I, S63L, L91M, R119L, K268R, T311G, E339V, and I353S;
(e) E59N, S63L, L91M, R119A, K268R, T311G, E339V, and I353S; and
(f) E59I, S63L, L91M, R119A, K268R, T311G, E339V, and I353S.
32. The method of any one of claims 27-31, wherein the enzyme further comprises a heterologous peptide fragment.
33. The method of claim 32, wherein the heterologous peptide fragment is an XTEN polypeptide, an IgG Fc, albumin, an albumin binding peptide, or polysialic acid time extension.
34. The method of any one of claims 27-31, wherein the enzyme is conjugated to polyethylene glycol (PEG).
35. The method of claim 34, wherein the enzyme is coupled to PEG through one or more lysine or cysteine residues.
36. The method of claim 27, wherein the subject is maintained on a methionine restricted diet.
37. The method of claim 27, wherein the subject maintains a normal diet.
38. The method of claim 27, wherein the subject is a human patient.
39. The method of claim 27, wherein the formulation is administered intravenously, intraarterially, intraperitoneally, intralesionally, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intravitreally, intramuscularly, intravesicularly, intraumbilically, by injection, infusion, continuous infusion, direct local perfusion bathing target cells, or via a catheter.
40. The method of claim 27, wherein the subject has previously been treated for homocystinuria or hyperhomocysteinemia and the enzyme is administered to prevent the recurrence of the homocystinuria or hyperhomocysteinemia.
41. The method of claim 27, further comprising administering to the subject at least a second homocystinuria or hyperhomocysteinemia therapy.
42. The method of claim 41, wherein said second homocystinuria or hyperhomocysteinemia therapy is high dose vitamin B6 or betaine (N, N, N-trimethylglycine) therapy.
43. The therapeutic preparation according to claim 25 for use as a medicament for treating a subject suffering from homocysteinuria or hyperhomocysteinemia.
44. The enzyme of any one of claims 1-14 for use in treating homocysteinuria or hyperhomocysteinemia in a subject.
As used herein, the term "encoding" or "encoding" with respect to a nucleic acid is used to facilitate understanding of the present invention by those skilled in the art. However, these terms may be used interchangeably with "comprising" or "comprising", respectively.
As used herein, "substantially free" with respect to a particular component is used herein to mean that any particular component is not intentionally formulated into a composition and/or is present only as a contaminant or trace amount. Thus, the total amount of a particular component resulting from any accidental contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions that cannot detect a particular component using standard analytical methods.
As used herein, "a" or "an" may refer to one or more. As used in one or more claims, "a" or "an" when used in conjunction with the word "comprising" may mean one or more.
Although the present disclosure supports the definition of substitute and "and/or" only, the term "or" as used in the claims means "and/or" unless it is expressly stated that only a reference to a substitute or a substitute is mutually exclusive. "another", as used herein, may mean at least a second or more.
As used herein, the term "about" is understood by one of ordinary skill in the art and will vary to some extent in the context in which it is used. Generally, a range of values including plus/minus 10% of the reference value is included.
Brief description of the drawings
The following figures form part of the present specification and are included to further illustrate the methods, compounds, and compositions.
FIG. 1-evaluation of modified CGL in vivo. The enzyme was administered in a single dose of 50mg/kg (i.p.). Mutant 1 corresponds to hCGL-NLV (SEQ ID NO:2), and mutant 2 corresponds to hCGL-8mut-4(SEQ ID NO: 6). Serum was collected for one week before injection and every 24 hours after injection.
Figure 2-multidose pharmacodynamics. The enzyme (hCGL-8 mut-4; SEQ ID NO:6) was administered at 50mg/kg (i.p.) every 5 days. Serum was collected before and 24 hours after each injection.
Figure 3-in vivo therapeutic effect of modified CGL. The enzyme (hCGL-8 mut-4; SEQ ID NO:6) was injected twice a week starting on postnatal day 10. The top line represents the modified CGL. The bottom line represents inactive enzyme.
Figure 4-evaluation of mutant 3 in vivo. The enzyme (Mutant 3; SEQ ID NO:37) was administered once at a dose of 50mg/kg (i.p.). Serum was collected for one week before injection and every 24 hours after injection. The top line represents the inactivated enzyme. The bottom line represents mutant 3(SEQ ID NO: 37).
Figure 5-in vivo therapeutic effect of mutant 3. The enzyme (Mutant 3; SEQ ID NO:37) was injected twice a week starting on postnatal day 10.
FIG. 6-SEQ ID NO:1 and 7-10. Asterisks indicate the engineered positions in the various modified CGL enzymes.
Detailed Description
Methods of treating diseases (e.g., homocysteinuria and hyperhomocysteinemia) using modified therapeutic enzymes that degrade homocysteine and homocysteine are provided. Therapeutic enzymes include hCGL-NLV mutants (E59N, R119L, E339V) and recombinantly engineered human cystathionine-gamma-lyase (hCGL) with improved homotypic caspase activity relative to hCGL-NLV mutants. Mutants that exhibit higher catalytic activity require lower concentrations of therapeutic agent for patient administration.
I. Definition of
As used herein, the terms "enzyme" and "protein" and "polypeptide" refer to compounds comprising amino acids linked by peptide bonds, and are used interchangeably.
As used herein, the term "fusion protein" refers to a chimeric protein comprising a protein or protein fragment operably linked in a non-native manner.
As used herein, the term "half-life" (1/2-life) refers to the time required for the concentration of a polypeptide to decrease by half, e.g., after injection in a mammal, in vitro (e.g., as measured in cell culture medium) or in vivo (e.g., as measured in serum). The method of measuring the "half-life" includes using an antibody specific to CGL or PEG used in an ELISA (enzyme-linked immunosorbent assay) format in order to measure the physical amount of the protein according to time. Other methods closely related to measuring half-life include determining the change in catalytic activity of an enzyme drug over time by any detection method that can detect the production of any substrate due to the conversion of homotypic (cysteine) to a product (e.g., alpha-ketobutyrate, methyl mercaptan, and/or ammonia).
The terms "in operable combination," "in operable order," and "operably linked" refer to a linkage in which the components are in a relationship that allows them to function in their intended manner, e.g., nucleic acid sequences are linked in a manner that is capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule, or amino acid sequences are linked in a manner that produces a fusion protein.
The term "linker" refers to a compound or moiety that acts as a molecular bridge to operably link two different molecules, wherein one portion of the linker is operably linked to a first molecule, and wherein another portion of the linker is operably linked to a second molecule.
The term "pegylation" refers to conjugation to polyethylene glycol (PEG), which is widely used as a drug carrier due to its high degree of biocompatibility and ease of modification. PEG can be chemically coupled (e.g., covalently linked) to the active agent via hydroxyl groups at the ends of the PEG chain; however, PEG itself is limited to only a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been investigated as novel biomaterials that would retain the biocompatibility of PEG, but have the additional advantage of many attachment points per molecule (thus providing greater drug loading) and can be designed synthetically to suit a variety of applications.
The term "gene" refers to a DNA sequence that comprises the control and coding sequences necessary for the production of a polypeptide or a precursor thereof. The polypeptide may be encoded by the full length coding sequence or any portion of the coding sequence so as to retain the desired enzymatic activity.
The term "native" refers to a typical or wild-type form of a gene, gene product, or a characteristic of the gene or gene product when isolated from a naturally occurring source. In contrast, the terms "modified," "variant," "mutein" or "mutant" refer to a gene or gene product that exhibits modified sequence and functional properties (i.e., altered characteristics) when compared to the native gene or gene product, wherein the modified gene or gene product is genetically engineered and does not occur in nature.
The term "vector" is used to refer to a vector nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell for replication. A nucleic acid sequence may be "exogenous," meaning that the sequence is foreign to the cell into which the vector is introduced or that the sequence is homologous to a sequence in the cell but is located in a host cell nucleic acid for which the nucleic acid sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well equipped to construct vectors by standard recombinant techniques (see, e.g., Maniatis et al, 1988 and Ausubel et al, 1994, both of which are incorporated herein by reference).
The term "expression vector" refers to any type of genetic construct, including nucleic acids encoding RNA that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, for example, during the production of antisense molecules or ribozymes, these sequences are not translated. Expression vectors can contain a variety of "control sequences" which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions.
As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic composition (i.e., a modified CGL enzyme or nucleic acid encoding such an enzyme) used in a method of achieving a therapeutic effect (i.e., depletion of homotypic (cysteine) in a patient's blood circulation to a level at or below a normal reference value). The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of a subject in the medical aspects of the present conditions. This includes, but is not limited to, a reduction in the frequency or severity of disease symptoms or signs, such as elevated (e.g., above 15 μmol/L) total homocysteine serum levels. The therapeutic composition should be administered in a dosage range large enough to produce the desired effect of reducing symptoms of homocysteinuria. For example, a therapeutically effective amount of a therapeutic composition may be an amount sufficient to achieve a concentration of intravascular (plasma) modified CGL enzyme of from about 0.001 to about 100U/mL, preferably greater than 0.1U/mL, and more preferably greater than 1.0U/mL when administered in a physiologically tolerable composition. Typical dosages may be administered on a weight basis, ranging from about 1 to about 100U/kg/day, preferably from about 2 to about 25U/kg/day, more preferably from about 2.0 to about 8.0U/kg/day. A typical amount may be 5.0U/kg/day or 35U/kg/week. The dosage should not be too large to cause adverse side effects such as ultrahigh viscosity syndrome, pulmonary edema, congestive heart failure, etc. In general, the dosage can be determined by one skilled in the art according to the age, symptoms, sex and condition of the patient. The dosage can also be adjusted by the physician in the event of complications. The dose should result in a reduction of the subject's serum tHcy level of at least 50%, at least 60%, and at least 70% within about 12 to 24 hours. The dose results in a reduction in serum tHcy levels in the subject of at least 50% to 70% within 6 hours. The dose which produces a statistically significant response should cause the subject serum tHcy level to be reduced to a level within two standard deviations of the reference level published by Nygard et al (1998). Thus, the dose may result in a reduction of the tHcy content in the serum of the subject to a level below 20-25 μ M.
The term "K" as used hereinM"refers to the Michaelis-Menten constant of an enzyme, defined as the concentration of a particular substrate at which a given enzyme produces half its maximum rate in a catalytic reaction. The term "k" as used hereincat"refers to the number of revolutions per substrate molecule converted per enzyme site to product per unit time, and wherein the enzyme operates at maximum efficiency. The term "k" as used hereincat/KM"refers to the specificity constant, which is a measure of the efficiency of an enzyme to convert a substrate to a product.
The term "cystathionine-gamma-lyase" (CGL or cystathionase) refers to any enzyme that catalyzes the hydrolysis of cystathionine to cysteine. As used herein, the term also contemplates primate forms of cystathionine- γ -lyase, including human forms of cystathionine- γ -lyase.
"Treatment" or "treating" refers to administering or applying a therapeutic agent to a subject, or performing surgery or physical therapy on a subject, to obtain a therapeutic benefit for a disease or health-related condition. For example, treatment may comprise administration of a therapeutically effective amount of a homotype (cysteine) enzyme.
"subject" and "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. The subject may be a human.
Cystathionine-gamma-lyase
A lyase is an enzyme that catalyzes the cleavage of various chemical bonds, usually resulting in the formation of new double bonds or new cyclic structures. For example, the enzyme catalyzing the reaction should be a lyase: ATP → cAMP + PPi. Lyases differ from other enzymes in that they require only one substrate for the unidirectional reaction and two substrates for the reverse reaction.
Many of the 5' -phosphopyridoxal (PLP) -dependent enzymes are involved in the metabolism of cysteine, homocysteine and methionine, and these enzymes form an evolutionarily related family known as Cys/Met metabolism PLP-dependent enzymes. These enzymes are proteins consisting of about 400 amino acids, with the PLP group attached to a lysine residue located at the central position of the polypeptide. Members of this family include cystathionine-gamma-lyase (CGL), cystathionine-gamma-synthase (CGS), cystathionine-beta-lyase (CBL), methionine-gamma-lyase (MGL), and O-acetylhomoserine (OAH)/O-acetyl-serine (OAS) sulfhydrylase (OSHS). All of them share the common feature of forming the mie complex, thereby forming the exo-substrate aldimine. The further course of the reaction is determined by the substrate specificity of the particular enzyme.
For example, the inventors introduced specific mutations into PLP-dependent lyase family members (such as human cystathionine- γ -lyase) to alter their substrate specificity. In this way, the present inventors produced novel variants with de novo synthesis capability to degrade homotypic cystine as substrate with a catalytic activity higher than that of hGGL-NLV. It is also contemplated that other PLP-dependent enzymes may be modified to produce novel homotypic (cysteine) degrading activities.
CGL is a tetramer that catalyzes the last step of the mammalian sulfur transfer pathway (Rao et al, 1990). CGL catalyzes the conversion of L-cystathionine to L-cysteine, alpha-ketobutyric acid and ammonia. Pyridoxal phosphate is the prosthetic group of this enzyme. Cystathionase, which has only weak degradation activity to homocysteine and homocysteine, is converted into an enzyme capable of degrading homocysteine and homocysteine at high speed using protein engineering (described in U.S. patent No. 9,481,877, which is incorporated herein by reference in its entirety).
Homotypic (cysteine) enzyme engineering
Since homo (cysteines are not produced by humans, there is a need to engineer homo (cysteines) for human therapy to have high activity and specificity for degrading homo (cysteines) under physiological conditions, as well as high stability in physiological fluids, such as serum, and to be generally non-immunogenic, since they are natural proteins that normally cause immune tolerance.
Since undesirable immunogenic effects were observed in animal studies with pMGL (MGL from pseudomonas putida), it was necessary to engineer the homo-type (cysteine) degrading activity of the human enzyme. The immunological tolerance to human proteins makes this enzyme potentially non-immunogenic or minimally immunogenic and therefore well tolerated.
Although mammals do not have homotypic (cysteine) caspases, they do have cystathionine-gamma-lyase (CGL). CGL is a tetramer that catalyzes the last step of the mammalian sulfur transfer pathway (Rao et al, 1990). CGL catalyzes the conversion of L-cystathionine to L-cysteine, alpha-ketobutyric acid and ammonia. Human CGL (hCGL) cDNA has been previously cloned and expressed, but the yield is relatively low (about 5mg/L culture) (Lu et al, 1992; Steegborn et al, 1999).
Thus, methods and compositions related to primate (particularly human) cystathionine- γ -lyase (CGL or cystathionase) modified by mutagenesis to efficiently hydrolyze homotypic (cysteine) are provided.
Modified CGL enzymes are described which exhibit at least one functional activity comparable to that of the unmodified CGL enzyme. The modified CGL enzyme may be further modified to improve serum stability. Modified CGL enzymes include, for example, proteins with additional advantages, such as homotypic (cysteine) enzyme activity, compared to unmodified CGL enzymes. The unmodified protein or polypeptide may be a native cystathionine- γ -lyase, such as human cystathionine- γ -lyase.
The determination of the activity, in particular with respect to the enzymatic activity, can be effected by detection methods familiar to the person skilled in the art and may comprise, for comparison purposes, for example, the use of natural and/or recombinant forms of modified or unmodified enzymes. For example, homotypic (cysteine) enzyme activity may be determined by any assay that detects any substrate produced by conversion of homotypic (cysteine), such as alpha-ketobutyrate, methionine and/or ammonia.
Modified CGL enzymes can be identified based on their enhanced homotypic (cysteine) degradation activity. For example, a substrate recognition site of an unmodified polypeptide can be recognized. This identification can be based on structural analysis or homology analysis. It is possible to generate a population of mutants comprising modifications to such substrate recognition sites. Mutants having enhanced homotypic (cysteine) degradation activity may be selected from this population of mutants. Selection of the desired mutant may include methods such as detection of by-products or products from degradation of homotypic (cysteine).
The modified CGL enzyme may have amino acid deletions and/or substitutions; thus, enzymes with deletions, enzymes with substitutions, enzymes with deletions and substitutions are all modified CGL enzymes. These modified CGL enzymes may further include inserted or added amino acids, such as fusion proteins or linker proteins. A "modified deletion CGL enzyme" lacks one or more residues of the native enzyme, but may have the specificity and/or activity of the native enzyme. Modified deletion CGL enzymes may also reduce immunogenicity or antigenicity. An example of a modified deleted CGL enzyme is an enzyme having one amino acid residue deleted from at least one antigenically active region that is the region of the enzyme identified as being antigenically active in a particular organism, such as the type of organism to which the modified CGL enzyme may be administered.
Substitution or substitution variants may comprise the exchange of one amino acid for another at one or more sites within a protein, and may be designed to modulate one or more properties of the polypeptide, particularly its effector function and/or bioavailability. Substitutions may or may not be conservative, i.e., an amino acid is substituted with an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.
In addition to deletions or substitutions, the modified CGL enzymes may have an insertion of a residue, which typically involves the addition of at least one residue in the enzyme. This may include the insertion of a targeting peptide or polypeptide or simply a single residue. Terminal additions (also referred to as fusion proteins) will be discussed below.
The term "biofunctional equivalent" is well understood in the art and is defined herein in further detail. Thus, including CGL enzyme sequences having about 90% or more sequence identity to SEQ ID NO 1, or amino acids that are identical or conservatively substituted with amino acids even from about 91% to about 99% (including 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%) of the modified CGL enzymes disclosed herein, results in measurable parameters of biological activity (e.g., the conversion of homotypic (cysteine) to alpha-ketobutyrate, methionine and ammonia) within about 20%, about 15%, about 10% or about 5% of the modified CGL enzymes disclosed herein, provided that the biological activity of the enzyme is maintained. The modified CGL enzyme may be biologically functionally identical to the unmodified CGL enzyme.
It will also be appreciated that the amino acid and nucleic acid sequences may include additional residues (such as additional N-or C-terminal amino acids or 5 'and 3' sequences), however, these sequences remain essentially one of the sequences disclosed herein as previously described, so long as the sequences meet the standard requirements set forth above, including maintaining biological protein activity in terms of protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences which, for example, may include various non-coding sequences flanking either the 5 'or 3' portion of the coding region, or may include various internal sequences known to be present in the gene, i.e., introns.
Specifically, five amino acid positions were found to be identical to hCGL-NLV (SEQ ID NO:2) except for three mutation sites (i.e., E59N-R119L-E339V). These additional positions are located at residues 63, 91, 268, 311, 353 of hCGL (SEQ ID NO:1) (see FIG. 6). One or more of these positions was mutated at S63L, L91M, K268R, T311G and I353S, and in combination with mutations at residues 59, 119 and 339, the activity was increased compared to hCGL-NLV. In particular, variants comprising a nucleic acid sequence corresponding to SEQ ID NO 3, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8 mut-1); 4, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8 mut-2); 5, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8 mut-3); 6, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8 mut-4); 27, hCGL-E59N-R119L-T336D-E339V (hCGL-NLDV); 28, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-1); 29, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-2); 30, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-3); 31, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9 mutD-4); 32, hCGL-E59N-R119L-T336E-E339V (hCGL-NLEV); 33, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-1); 34, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-2); 35, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-3); 36, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9 mutE-4); 37, hCGL-E59I-S63L-L91M-R119D-K268R-T311G-E339V-I353S (mutant 3); 38, hCGL-E59I-S63L-L91M-R119H-K268R-T311G-E339V-I353S (mutant 4); and amino acid substitutions of SEQ ID NO 39, hCGL-E59I-S63L-L91M-R119G-K268R-T311G-E339V-I353S (mutant 5).
Enzymatic homo-cystine depletion for therapy
The polypeptides are useful in the treatment of diseases (such as homocysteinuria) by depleting homocystine and/or homocysteine novel enzymes. Methods of treatment using modified CGLs that contain L- (cysteine degrading activity are disclosed. Enzymes having homotypic (cysteine) degrading activity are provided to enhance therapeutic efficacy.
Modified CGL enzymes having homotypic cystine degrading activity are provided for use in treating diseases such as homocystinuria. In particular, the modified polypeptide may have a human polypeptide sequence, and thus, when administered to a human patient, it may prevent adverse immunogenic reactions, allow for repeated administration, and improve therapeutic efficacy.
The depletion may be performed in the following cases: in vitro in blood circulation in a mammal, in the case of tissue culture or other biological media requiring homotypic cystine deficiency, in vitro manipulation of biological fluids, cells or tissues and subsequent return to the mammal patient's body ex vivo. Depletion of homo (cysteine) is performed in blood circulation, culture medium, biological fluids, or cells to reduce the amount of homo (cysteine) available from the material being treated, and therefore it involves contacting the material to be depleted with a homo (cysteine-depleting amount of an engineered enzyme under homo (cysteine-depleting conditions to degrade the environmental homo (cysteine) in the contacted material.
The efficiency of homotypic cystine depletion varies greatly from application to application and generally depends on the amount of homotypic (cysteine) present in the material, the desired rate of depletion and the tolerance of the material to exposure to homotypic (cysteine). The level of homotypic (cysteine) in a material and the rate of depletion of homotypic (cysteine) from that material can be readily monitored by a variety of chemical and biochemical methods well known in the art. Further described herein are exemplary homo-type cystine depletion amounts that range from 0.001 to 100 units (U), preferably between about 0.01 and 10U, more preferably between about 0.1 and 5U, of engineered enzyme per milliliter (mL) of material to be treated.
Homo (cysteine) depleting conditions are buffering and temperature conditions compatible with the biological activity of the homo (cysteine) enzyme, including mild temperature, salt and pH conditions compatible with the enzyme, such as physiological conditions. Exemplary conditions include about 4-40 ℃, an ionic strength of about 0.05-0.2M NaCl, a pH of about 5-9, and physiological conditions.
The contacting in vivo may be accomplished by administration, intravenous or intraperitoneal injection, and the therapeutically effective amount of the physiologically tolerable composition comprises the modified CGL enzyme administered to the patient, thereby depleting homotypic (cysteine) in the patient's blood circulation.
The modified CGL enzyme may be administered parenterally by injection or gradual infusion. The modified CGL enzyme may be administered intravenously, intraperitoneally, intramuscularly, may be injected directly into the tissue containing the tumor cells, or may also be administered via a pump connected to a catheter, which may contain a potential biosensor or homocystine.
Therapeutic compositions containing the modified CGL enzyme are typically administered intravenously, e.g., by injection of a unit dose. The term "unit dose" when used in reference to a therapeutic composition, refers to physically discrete units suitable for use in a subject as unitary dosages, each unit containing a predetermined quantity of active material which causes the desired therapeutic effect in association with the required diluent (i.e., carrier or vehicle).
The composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount administered will depend upon the subject to be treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient to be administered depends on the judgment of the practitioner and is specific to each individual. However, dosage ranges suitable for systemic use are also disclosed herein, and will depend on the route of administration. Also contemplated are regimens suitable for initial administration and booster injections, which are typically administered first, then repeated every hour or more, followed by injections or other routes of administration. Exemplary multiple administrations are described herein, particularly recommended to maintain consistently high serum and tissue levels of the modified CGL enzyme, whereas homotypic (cysteine) levels are maintained at low serum and tissue levels. Alternatively, continuous intravenous infusion may be considered to maintain the concentration in the blood within the in vivo therapeutic range. Notably, the weekly dose of the modified CGL enzyme for the treatment of homocysteinuria and hyperhomocysteinemia is about one-fourth of the weekly dose required for cancer treatment. U.S. patent No. 9,481,877.
V. conjugates
The compositions and methods provided involve further modification of the modified CGL enzyme to improve, for example, by forming conjugates with heterologous peptide fragments or polymers such as polyethylene glycol. The modified CGL enzyme can be linked to PEG to increase the hydrodynamic radius of the enzyme, thereby increasing serum persistence. The disclosed polypeptides may be coupled to any targeting agent, such as a ligand capable of specifically and stably binding to an external receptor or binding site of a tumor cell (U.S. patent publication 2009/0304666). The PEG is between about 3,000 and 20,000 daltons in size, with an exemplary size being 5,000 daltons.
A. Fusion proteins
In fusion proteins, the modified CGL enzyme may be linked to a heterologous domain at the N-or C-terminus. For example, fusion may also use leader sequences from other species to allow recombinant expression of the protein in a heterologous host. Another useful fusion includes the addition of a protein affinity tag (such as a serum albumin affinity tag or hexahistidine residues) or an immunologically active domain (such as an antibody epitope, preferably cleavable) to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, Chitin Binding Protein (CBP), Maltose Binding Protein (MBP), and glutathione-s-transferase (GST).
The modified CGL enzyme may be linked to a peptide that increases half-life in vivo, such as an XTEN polypeptide (Schellenberger et al, 2009), IgG Fc domain, albumin or albumin binding peptide.
Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of a complete fusion protein, or by ligation of DNA sequences encoding heterologous domains and expression of the complete fusion protein.
The production of fusion proteins that restore the functional activity of the parent protein can be facilitated by linking the gene to a bridging DNA segment that encodes a peptide linker that is spliced between the polypeptides linked in tandem. The linker should be of sufficient length to allow proper folding of the resulting fusion protein.
B. Joint
The modified CGL enzyme may be chemically conjugated with a bifunctional cross-linking reagent or fused at the protein level to a peptide linker. Bifunctional cross-linking reagents have been widely used for a variety of purposes, including the preparation of affinity matrices, the modification and stabilization of various structures, the recognition of ligand and receptor binding sites, and structural studies. Suitable peptide linkers (such as Gly-Ser linkers) may also be used to link the modified CGL enzyme.
Homologous bifunctional reagents carrying two identical functional groups can cause cross-linking between the same or different macromolecules or macromolecular subunits and link the polypeptide ligand to its specific binding site. The heterobifunctional reagent comprises two different functional groups. The crosslinking can be controlled selectively and sequentially by virtue of the different reactivity of the two different functional groups. Bifunctional crosslinking reagents can be classified into amino-, mercapto-, guanidino, indolyl, carboxyl-specific groups, and the like, depending on the specificity of the functional group. Among them, reagents directed to free amino groups are favored for their commercial availability, ease of synthesis, and suitable mild reaction conditions.
Some heterobifunctional crosslinking reagents contain one primary amine-reactive group and a thiol-reactive group. In another example, a heterologous bifunctional crosslinking reagent and methods of using the crosslinking reagent are described (U.S. patent No. 5,889,155, which is more specifically incorporated herein by reference in its entirety). The crosslinking reagent binds nucleophilic hydrazide residues to electrophilic maleimide residues, allowing, for example, the coupling of aldehydes to free thiols. The crosslinking reagent can be modified to crosslink various functional groups.
In addition, any other linking/coupling agent and/or mechanism known to those skilled in the art may be used to bind the modified CGL enzyme, such as antibody-antigen interactions, avidin-biotin bonds, amide bonds, ester bonds, thioester bonds, ether bonds, thioether bonds, phosphate ester bonds, phosphoamide bonds, anhydride bonds, disulfide bonds, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.
It is preferred to use cross-linking agents with reasonable blood stability. Many types of disulfide bond containing linkers are known to be successfully used for conjugated targeting and therapeutic/prophylactic agents. Linkers containing sterically hindered disulfide bonds may be more stable in vivo. Thus, these linkers are a group of linkers.
In addition to hindered crosslinkers, non-hindered crosslinkers can also be used as specified herein. Other useful (not believed to contain or create protected disulfide bonds) cross-linking agents include SATA, SPDP and 2-iminothioether (Wawrzynczak and Thorpe, 1987). The use of such cross-linking agents is well understood in the art. Flexible joints may also be used.
Once chemically conjugated, the peptide is typically purified to separate the conjugate from the non-conjugating agent and other contaminants. A number of purification techniques can be used to provide conjugates of sufficient purity to make them clinically useful.
Purification methods based on particle size separation (such as gel filtration, gel permeation or high performance liquid chromatography) are generally the most common. Other chromatographic techniques such as Blue-Sepharose separation may also be used. Conventional methods for purifying fusion proteins from inclusion bodies may be useful, such as using weak detergents, such as Sodium Lauryl Sarcosinate (SLS).
C pegylation
Methods and compositions relating to pegylation of modified CGL enzymes are disclosed. For example, the modified CGL enzyme may be pegylated according to the methods disclosed herein.
Pegylation is the process of covalently linking a poly (ethylene glycol) polymer chain to another molecule, usually a drug or therapeutic protein. Pegylation is typically achieved by incubating a reactive derivative of PEG with the target macromolecule. Covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent against the host immune system (reduced immunogenicity and antigenicity), increase the hydrodynamic size of the agent (size in solution), which can prolong its circulation time by reducing renal clearance. Pegylation can also provide water solubility to hydrophobic drugs and proteins.
The first step of pegylation is the appropriate functionalization of the PEG polymer at one or both termini. PEG activated at each end with the same reactive moiety is referred to as "homobifunctional", whereas PEG derivatives are referred to as "heterobifunctional" or "heterofunctional" if the functional groups present are different. Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to the desired molecule.
The selection of suitable functional groups for PEG derivatives is based on the type of reactive groups available on the molecule to be coupled to PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids may also be used.
Techniques for forming first generation PEG derivatives typically react PEG polymers with groups that can react with hydroxyl groups (typically anhydrides, acid chlorides, chloroformates, and carbonates). In second generation pegylation chemistry, more potent functional groups (such as aldehydes, esters, amides, etc.) can be used for conjugation.
As the use of pegylation has become more advanced and complex, the demand for heterologous bifunctional PEG for conjugation has increased. These heterobifunctional PEGs are well suited for linking two entities where hydrophilic, flexible and biocompatible spacers are required. Preferred end groups for the heterobifunctional PEG are maleimide, vinyl sulfone, dithiopyridine, amines, carboxylic acids and NHS esters.
The most common modifying agents or linkers are based on methoxy peg (mpeg) molecules. Its activity depends on the addition of a protein modifying group to the alcohol terminus. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is then modified at both termini to produce a hetero-or homo-dimeric PEG linker molecule.
Proteins are typically pegylated at nucleophilic sites, such as unprotonated thiol (cysteinyl residues) or amino groups. Examples of cysteinyl specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiol, and PEG vinylsulfone. All four had strong cysteinyl specificity under mild conditions and neutral to slightly basic pH, but each had some drawbacks. The thioether formed by maleimide may be slightly less stable under alkaline conditions, and thus formulation options using this linker may have some limitations. The thiocarbamate linkage formed by iodopeg is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiols form disulfide bonds with protein thiols, but such bonds can also be unstable under basic conditions. PEG-vinyl sulfone reactivity is relatively slow compared to maleimide and iodopeg; however, the thioether bond formed is very stable. Its slower reaction rate also makes the PEG-vinyl sulfone reaction easier to control.
Site-specific pegylation at native cysteinyl residues is rarely performed, as these residues are usually in the form of disulfide bonds or are required for biological activity. In another aspect, site-directed mutagenesis can be used to incorporate a cysteinyl pegylation site for the thiol-specific linker. The cysteine mutation must be designed so that it is accessible to the pegylation agent and still biologically active after pegylation.
Amine-specific modifiers include PEG NHS esters, PEG trifluoroethylsulfonate, PEG aldehydes, PEG isothiocyanates, and the like. All of which react under mild conditions and are very specific for amino groups. PEG NHS ester may be a more reactive reagent; however, its high reactivity may make pegylation difficult to control on a large scale. PEG aldehydes form imines with amino groups, which are subsequently reduced to secondary amines with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride will not reduce the disulfide bond. However, this chemical is highly toxic and must be handled with caution, particularly at the lower pH where it is volatile.
Site-specific pegylation is challenging due to the multiple lysine residues on most proteins. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct pegylation to lower pK amino groups by conducting the reaction at lower pH. In general, the pK of the α -amino group is 1-2 pH units less than the ε -amino group of a lysine residue. By pegylating the molecule at pH7 or less, high selectivity to the N-terminus is generally obtained. However, this is only possible if the N-terminal part of the protein is not required for biological activity. In addition, the pharmacokinetic benefit of pegylation is generally greater than the significant loss of biological activity in vitro, resulting in a product with greater biological activity in vivo, regardless of pegylation chemistry.
A number of parameters need to be considered when developing the pegylation procedure. Fortunately, the parameters typically do not exceed four or five. "design of experiments" methods to optimize pegylation conditions are very useful. For thiol-specific pegylation, parameters to be considered include: protein concentration, PEG to protein ratio (on a molar basis), temperature, pH, reaction time, and in some cases oxygen removal. (oxygen can help achieve intermolecular disulfide formation by the protein, which will reduce the yield of pegylated products.) the same factors (other than oxygen) should be considered for amine-specific modification, pH may be even more critical, especially when targeting the N-terminal amino group.
With respect to amine and thiol specific modifications, reaction conditions can affect the stability of the protein. This can limit temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before the pegylation reaction is initiated. For example, if the pegylation agent is only 70% active, the amount of PEG used should ensure that only active PEG molecules are calculated in the protein to PEG reaction stoichiometry.
Proteins and peptides
Compositions comprising at least one protein or peptide, such as a modified CGL enzyme, are provided. These peptides may be included in the fusion protein or conjugated to the agents described above.
As used herein, a protein or peptide generally refers to, but is not limited to, a protein of greater than about 200 amino acids up to the full-length sequence translated from the gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.
Thus, the term "protein or peptide" encompasses proteins comprising at least one of the 20 common amino acids in naturally occurring proteins, or at least one modified or unnatural amino acid.
The protein or peptide may be prepared by any technique known to those skilled in the art, including expression of the protein, polypeptide, or peptide by standard molecular biology techniques, isolation of the protein or peptide from a natural source, or chemical synthesis of the protein or peptide. Coding regions for known genes may be amplified and/or expressed using techniques disclosed herein or known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those skilled in the art.
Nucleic acids and vectors
Nucleic acid sequences encoding modified CGLase or fusion proteins containing modified CGLase are disclosed. The nucleic acid sequence may be selected on the basis of conventional methods, depending on the expression system used. For example, if the modified CGL enzyme is derived from human cystathionase and contains multiple codons that are rarely used in E.coli, it may interfere with expression. Thus, coding sequences without rare codons can be designed using toll-free software (see Hoover & Lubkowski, 2002) for codon optimization of individual genes or variants thereof for e. Various vectors may also be used to express the protein of interest, such as a modified CGL enzyme. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.
VIII. host cell
The host cell may be any cell that can be transformed to allow expression and secretion of the modified CGL enzyme and its conjugate. The host cell may be a bacterium, a mammalian cell, a yeast or a filamentous fungus. Various bacteria include the genera Escherichia (Escherichia) and Bacillus (Bacillus). Yeasts belonging to the genera Saccharomyces (Saccharomyces), Kluyveromyces (Kiuyveromyces), Hansenula (Hansenula) or Pichia (Pichia) will be useful as suitable host cells. Various species of filamentous fungi may be used as expression hosts, including the following genera: aspergillus (Aspergillus), Trichoderma (Trichoderma), Neurospora (Neurospora), Penicillium (Penicillium), Cephalosporium (Cephalosporium), Achlya (Achlya), Podospora (Podospora), Endothia (Endothia), Mucor (Mucor), Cochlospora (Cochliobolus), and Pyricularia (Pyricularia).
Examples of useful host organisms include bacteria such as Escherichia coli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al, 1984), Staphylococcus aureus (Staphylococcus aureus) SAI123 (Lordanesu, 1975), or Streptococcus lividans (Streptococcus lividans) (Hopwood et al, 1985); yeasts, for example Saccharomyces cerevisiae AH 22(Mellor et al, 1983) or Schizosaccharomyces pombe (Schizosaccharomyces pombe); and filamentous fungi, such as Aspergillus nidulans (Aspergillus nidulans), Aspergillus awamori (Ward, 1989) or Trichoderma reesei (Trichoderma reesei) (Penttila et al, 1987; Harkki et al, 1989).
Examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1; American Type Culture Collection (ATCC) No. CCL61), rat pituitary cells (GH 1; ATCC No. CCL82), Hela S3 cells (ATCCno. CCL2.2), rat hepatoma cells (H-4-II-E; ATCC No. CRL-1548), SV40 transformed monkey kidney cells (COS-1; ATCC No. CRL-1650), and murine blastocytes (NIH-3T 3; ATCC No. CRL-1658). The foregoing is illustrative, and not limiting, of the many possible host organisms known in the art. In principle, all hosts capable of secretion can be used, whether prokaryotic or eukaryotic.
The mammalian host cells expressing the modified CGL enzyme and/or fusion protein thereof are cultured under conditions typically used to culture the parental cell line. Generally, cells are cultured in standard medium (such as standard RPMI, MEM, IMEM, or DMEM) containing physiological salts and nutrients, typically supplemented with 5% -10% serum (such as fetal bovine serum). Culture conditions are also standard, e.g., cultures are incubated at 37 ℃ in static or rotary cultures until the desired protein level is achieved.
IX. protein purification
Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, homogenization and crude separation of cells, tissues or organs into polypeptide and non-polypeptide fractions. Unless otherwise specified, the protein or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. A particularly effective method for purifying peptides is fast high performance liquid chromatography (FPLC) or even High Performance Liquid Chromatography (HPLC).
By purified protein or peptide is meant a composition that can be separated from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtained state. Thus, an isolated or purified protein or peptide also refers to a protein or peptide that is isolated from the environment in which it may naturally occur. In general, "purified" will refer to a protein or peptide composition that has been fractionated to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this name will refer to a composition in which the protein or peptide forms the major component of the composition, such as the protein comprising about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition.
Various techniques suitable for protein purification are well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc., or by heat denaturation followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. The order in which the various purification steps are performed may be varied, or certain steps may be omitted, and still be a suitable method for preparing a substantially purified protein or peptide, as is generally known in the art.
Various methods for quantifying the degree of purification of a protein or peptide are known to those skilled in the art. These methods include, for example, determining the specific activity of the active fraction, or assessing the amount of polypeptide within the fraction by SDS/PAGE analysis. A preferred method for analyzing the purity of a fraction is to calculate the specific activity of the fraction, compare it with the specific activity of the initial extract, and thereby calculate the degree of purity therein, which is evaluated by "fold purification". The actual unit used to express the amount of activity will, of course, depend on the particular detection technique chosen for purification, and whether the expressed protein or peptide exhibits detectable activity.
It is not generally required that the protein or peptide is always provided in its purest state. Indeed, it is contemplated that a substantially less pure product may have utility. Partial purification can be achieved by using fewer purification steps in combination or by using different forms of the same general purification scheme. For example, it is to be understood that cation exchange column chromatography using an HPLC apparatus typically results in a "fold" of purification that is greater than the same technique using a low pressure chromatography system. Methods with lower relative degrees of purification can have advantages in terms of overall recovery of the protein product, or in terms of maintaining the activity of the expressed protein.
The protein or peptide may be isolated or purified, for example a modified CGL enzyme, a fusion protein containing a modified CGL enzyme or a modified CGL enzyme after pegylation. For example, a His-tag or affinity epitope may be included in such modified CGL enzymes to facilitate purification. Affinity chromatography is a chromatographic procedure that relies on the specific affinity between the substance to be separated and the molecules to which it can specifically bind. This is a type of receptor-ligand interaction. Column materials are synthesized by covalently coupling one binding partner to an insoluble matrix. The column material is then capable of specifically adsorbing substances from the solution. Elution is performed by changing the conditions to those under which no binding occurs (e.g., altered pH, ionic strength, temperature, etc.). The matrix should be a substance that does not adsorb molecules to a large extent and has a wide range of chemical, physical and thermal stability. The ligands should be coupled in such a way that their binding properties are not affected. The ligand should also provide relatively tight binding. It should be possible to elute the material without destroying the sample or the ligand.
Size Exclusion Chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size or in a more technical term, hydrodynamic volume. It is generally applicable to macromolecules or macromolecular complexes, such as proteins and industrial polymers. Generally, this technique is referred to as gel filtration chromatography when an aqueous solution is used to transport a sample through a column, and gel permeation chromatography when an organic solvent is used as the mobile phase.
The basic principle of SEC is that particles of different sizes elute through the stationary phase at different rates (filtration). This separates the solution of particles on a size basis. If all particles are loaded simultaneously or nearly simultaneously, particles of the same size should elute together. Each size exclusion column has a separable molecular weight range. The exclusion limit limits molecular weight to the upper end of the range and is the limit where the molecules are too large to be captured in the stationary phase. The percolation limit limits the molecular weight to the lower end of the separation range where molecules of sufficiently small size can penetrate completely into the pores of the stationary phase and all molecules smaller than the molecular mass are too small to elute as a single band.
High performance liquid chromatography (or high pressure liquid chromatography, HPLC) is a form of column chromatography commonly used in biochemical and analytical chemistry to separate, identify and quantify compounds. HPLC utilizes a column containing a chromatographic packing material (stationary phase), a pump to move the mobile phase through the column, and a detector to show the retention time of the molecules. The retention time varies depending on the interaction between the stationary phase, the molecule being analyzed, and the solvent used.
Therapeutic compositions
The human cystathionine-gamma-lyase gene contains a plurality of codons which are rarely used in E.coli and which can interfere with expression. Thus, to optimize protein expression in E.coli, codon-optimized oligonucleotides designed by DNA-Works software can be used to assemble the corresponding genes (Hoover et al, 2002). Each construct may contain an N-terminal NcoI restriction site, an in-frame N-terminal His6 tag, and a C-terminal EcoRI site to simplify cloning. After cloning into pET28a vector (Novagen), E.coli (BL21) containing the appropriate cystathionase expression vector can be cultured in a medium containing 50. mu.g/mL kanamycin (TBTerrific Broth, TB) at 37 ℃ in a shake flask at 250rpm until OD is reached600-0.5-0.6. At this point, the culture can be transferred to a shaker at 25 ℃ and induced with 0.5mM IPTG and the protein re-expressed for 12 h. The cell pellet can then be collected by centrifugation and resuspended in IMAC buffer (10mM NaPO)410mM imidazole/300 mM NaCl, pH 8). After lysis by a French cell press, the lysate is centrifuged for 20min at 20,000x g at 4 ℃ and the resulting supernatant is applied to a nickel IMAC column, extensively washed with (90-100 column volumes) IMAC buffer containing 0.1% TRITONTM 114, washed with 10-20 column volumes IMAC buffer, and then eluted with IMAC elution buffer (50mM NaPO)4250mM imidazole/300 mM NaCl, pH 8). The purified protein can be buffer-liquor exchanged to 100mM NaPO using a 10,000MWCO (molecular weight cut-off) filtration device4The buffer pH 8.3. The enzyme containing fractions can then be incubated with 10mM PLP for 1h at 25 ℃. Methoxy PEG succinimidyl carboxymethyl ester 5000MW (JenKemTtechnology) may be added to CGL-8mut-1 at a molar ratio of 80:1 and allowed to react for 1h with constant stirring at 25 ℃. The resulting mixture was extensively buffer exchanged (10% glycerol in PBS) using a 100,000MWCO filtration unit (Amicon) and sterilized with a 0.2 micron syringe filter (VWR). Aliquots of the enzyme can be snap frozen in liquid nitrogen and stored at-80 ℃. The homogeneity of CGL or CGL variants purified in this manner may be assessed by SDS-PAGE and Coomassie stainingTo be provided with>95 percent. The extinction coefficient lambda can be obtained based on calculation under the conditions of final buffer solution concentration of 6M guanidine hydrochloride, 20mM phosphate buffer and pH value of 6.5280＝29870M-1cm-1The yield was calculated (Gill and von Hippel, 1989).
For example, the serum stability of pegylated hCGL-8mut-1 was tested by incubating the enzyme in collected human serum at a final concentration of 10. mu.M at 37 ℃. Aliquots were extracted and tested at various time points using the detection method of DTNB (Ellman's reagent; 5, 5-dithio-bis- (2-nitrobenzoic acid)), as described in U.S. patent publication 2011/0200576, which is incorporated herein by reference in its entirety. Half-life (T) of the resulting PEGylated hCGL-8mut-1 was calculated0.5) 101 + -4 hours.
There is no intention to limit the specific properties of the therapeutic formulation. For example, such compositions may be provided in formulations with physiologically tolerable liquid, gel or solid carriers, diluents and excipients. These therapeutic formulations can be administered to mammals for veterinary use, such as livestock, and to humans for clinical use in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary depending upon the type and mode of administration and the particular needs of the individual subject.
Such compositions are typically prepared as liquid solutions or suspensions for injection. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, and the like, and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffering agents.
Where clinical applications are contemplated, it may be desirable to prepare therapeutic compositions comprising proteins, antibodies and drugs in a form suitable for the intended application. In general, the pharmaceutical compositions may comprise an effective amount of one or more modified CGL variants or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The term "therapeutically or therapeutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when administered to an animal (e.g., a human), as the case may be. The preparation of pharmaceutical compositions containing at least one modified CGL enzyme, or another active ingredient, isolated by the methods disclosed herein will be known to those skilled in the art, as exemplified by Remington's pharmaceutical Sciences,18th edition, 1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it is understood that the formulations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA office of biological standards.
As used herein and known to those of ordinary skill in the art, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, adhesives, excipients, such similar materials, and combinations thereof (see, e.g., remington's pharmaceutical science, 18th edition, 1990, which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
The composition can be administered subcutaneously, intravenously, intraarterially, intraperitoneally, intramuscularly, by injection, infusion, continuous infusion, via catheter, in a lipid composition (e.g., liposomes), or by other methods as would be known to one of ordinary skill in the art, or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences,18th ed.,1990, which is incorporated herein by reference).
The modified polypeptides may be formulated into compositions in free base, neutral or salt form. Therapeutically acceptable salts include acid addition salts, such as those formed by free amino groups of the protein composition, or by inorganic (e.g., hydrochloric or phosphoric) or organic acids, such as acetic, oxalic, tartaric, or mandelic acid. Salts formed by free carboxyl groups may also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide); or an organic base such as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, the solution will be administered in a manner compatible with the dosage formulation and in such an amount as is therapeutically effective. The formulations may be administered in a variety of dosage forms, such as formulated for parenteral administration, such as an injection solution, or an aerosol for delivery to the lungs, or formulated for dietary administration, such as a drug release capsule or the like.
Compositions of the disclosure suitable for administration may be provided in a pharmaceutically acceptable carrier with or without an inert diluent. Unless any conventional media, agent, diluent or carrier is deleterious to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for practicing the method is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, and the like, or combinations thereof. The composition may also include various antioxidants to delay oxidation of one or more components. In addition, prevention of the action of microorganisms can be achieved by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methyl paraben, propyl paraben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.
The disclosed compositions are combined with a carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption, and the like.
There is provided the use of a therapeutic lipid vehicle composition comprising a CGL enzyme, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a wide range of substances characterized by being insoluble in water and extractable using organic solvents. This broad class of compounds is well known to those skilled in the art, and when the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids may be naturally occurring or synthetic (i.e., artificially designed or produced). However, lipids are typically biological substances. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, thioesters, lipids with ether and ester linked fatty acids, polymerizable lipids, and combinations thereof. Of course, the compositions and methods also encompass compounds other than those specifically described herein that are understood by those of skill in the art as lipids.
One of ordinary skill in the art will be familiar with the range of techniques that can be used to disperse the composition in a lipid vehicle. For example, the modified CGL enzyme or fusion protein thereof can be dispersed in a solution containing a lipid, solubilized with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently linked to a lipid, contained in suspension in a lipid, contained in or complexed with a micelle or liposome, or associated with a lipid or lipid structure by any means known to one of ordinary skill in the art. Dispersion may or may not cause liposome formation.
The actual dosage of the composition administered to an animal patient is determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic intervention, the patient's self-morbidity, and the route of administration. Depending on the dose and route of administration, the preferred dose and/or the number of administrations of the therapeutically effective amount may vary depending on the response of the subject. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject.
The therapeutic composition may comprise, for example, at least about 0.1% of the active compound. The active compound may comprise, for example, between about 2% and about 75%, or between about 25% and about 60%, by weight of the unit, and any range derivable therein. Of course, the amount of active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. One skilled in the art of preparing such pharmaceutical formulations will envision factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmaceutical considerations, and thus a variety of dosages and treatment regimens may be desirable.
The dose may also comprise about 500 micrograms/kg body weight, about 1 microgram/kg body weight, about 5 micrograms/kg body weight, about 10 micrograms/kg body weight, about 50 micrograms/kg body weight, about 100 micrograms/kg body weight, about 750 milligrams/kg body weight or more per administration, and any range derivable therein. If administered weekly, the dose may be 5mg/kg body weight, or for example, a 70kg subject may require 350mg of protein.
XI kit
Kits, such as therapeutic kits, can be provided. For example, a kit can comprise one or more of the therapeutic compositions described herein and optionally instructions for their use. The kit may further comprise one or more devices for effecting administration of such compositions. For example, a subject kit may comprise a therapeutic composition and a catheter for achieving intravenous injection of the composition directly into a target tissue. The kit may comprise pre-filled ampoules of modified CGL enzyme, optionally formulated as a therapeutic composition or lyophilized, for use with a delivery device.
The kit may comprise a container having a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container may contain a composition comprising a modified CGL enzyme effective for therapeutic or non-therapeutic applications as described above. The label on the container may indicate that the composition, such as described above, is for a particular therapeutic or non-therapeutic application, and may also indicate directions for in vivo or in vitro use. The kits of the present invention will generally comprise the container described above and one or more other containers containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
Xii example:
the following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.