Albumin-binding arginine deiminase and the use thereof

Abstract

The present invention provides a pharmaceutical composition containing albumin-binding arginine deiminase fusion protein (AAD) for treating cancer or other arginine-dependent diseases. The AAD fission protein can be purified from both soluble and insoluble fractions of crude proteins, it binds to human serum albumin (HSA) and has its high activity with longer half life for efficient depletion of arginine in cancer cells. The specific activities of wild-type ADI and AAD in the present invention are 8.4 and 9.2 U/mg (at physiological pH 7.4), respectively. The AAD used in the present invention can be used in the treatment of various cancers (e.g. pancreatic cancer, leukemia, head and neck cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, nasopharyngeal cancer, esophageal cancer, prostate cancer, stomach cancer & brain cancer) and curing arginine-dependent diseases. The composition can be used alone or in combination with at least one chemotherapeutic agent to give a synergistic effect on cancer treatment and/or inhibiting metastasis.

Description

CROSS-REFERENCE TO RELATED APPLICATION

The present application

This assay is run by adding sample to a color reagent, which is made by mixing acidic ferric chloride solution with DAM-TSC solution. Briefly, enzyme is incubated with 20 mM arginine, 10 mM sodium phosphate pH 7.4 for 5 min at 37° C. The reaction mixture is heated at 100° C. for 5 min to develop the color and read at 540 nm (light path=1 cm). A standard curve is constructed using various concentrations of citrulline. One unit of the ADI native enzyme is the amount of enzyme activity that converts 1 μmol of arginine to 1 μmol of citrulline per minute at 37° C. under the assay conditions. The specific activities of wild-type ADI and AAD fusion protein in the present invention are 8.4 and 9.2 U/mg (at pH 7.4, physiological pH) respectively. The specific activities for wild-type ADI and AAD fusion protein at different pH range (from pH 5.5 to 9.5) are also determined, and the optimum pH is at 6.5. Therefore, the results indicate that AAD fusion protein depletes arginine efficiently, as the fusion with albumin-binding protein does not affect enzyme activity of ADI.

The Michaelis constant K_m is the substrate concentration at which the reaction rate is at half-maximum, and is an inverse measure of the substrate's affinity for the enzyme. A small K_m indicates high affinity for the substrate, and it means that the rate will approach the maximum reaction rate more quickly. For determination of the enzyme kinetics or K_m value, the activity of wild-type ADI and AAD fusion protein are measured under different concentration of substrate arginine (2000 μM, 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM) at pH 7.4. The measured K_m values of the AAD fusion protein shown in FIG. 3E (SEQ ID NO: 40, ADI protein is originated from Mycoplasma arginini) and AAD fusion protein shown in FIG. 3F (SEQ ID NO: 41, ADI protein is originated from Bacillus cereus) are 0.0041 mM and 0.132 mM respectively. The results suggest that the fusion to ABD did not affect the binding affinity of the different AAD fusion proteins to arginine.

Example 5

Cell Proliferation Assay and In Vitro Efficacy of AAD Fusion Protein on Cancer Cell Lines

Culture medium DMEM is used to grow the human melanoma A375 & SK-mel-28, human pancreatic cancer PancI and human cervical cancer C-33A cell lines. The EMEM medium is used to culture the SK-hep 1 liver cancer and C-33A cervical cancer cell line. Cancer cells (2−5×10³) in 100 μl culture medium are seeded to the wells of 96-well plates and incubated for 24 h. The culture medium is replaced with medium containing different concentrations of AAD fusion protein. The plates are incubated for an additional 3 days at 37° C. in an atmosphere of 95% air/5% CO₂. MTT assay is performed to estimate the number of viable cells in the culture according to manufacturer's instructions. The amount of enzyme needed to achieve 50% inhibition of cell growth is defined as IC₅₀.

As shown in TABLE 1 and FIG. 9, the results indicate that AAD fusion protein depletes arginine efficiently and inhibits the growth of various types of human cancer cell lines in in vitro tissue culture studies. For example, human melanoma, human colon carcinoma, human pancreatic cancer, human liver cancer and human cervical cancer, all have low values of IC₅₀ (see TABLE 1), as these cancer types are all inhibited by AAD fusion protein readily. As predicted, AAD fusion protein would inhibit all cancer types that are arginine-dependent (for example, the ASS-negative cancers).

Example 6

In Vivo Half-Life Determination of AAD Fusion Protein

Balb/c mice (5-7 weeks) are used in this study and they are allowed to acclimatize for a week before the experiment. Mice (n=3) are separated into four groups and injected with 0, 100, 500 or 1000 μg of AAD fusion protein (SEQ ID NO: 40, FIG. 3E) in 100 μl PBS intraperitoneally, respectively. Blood of each mouse is collected at 0 h and Day 1-7. Sera are obtained after centrifugation. The sera are then deproteinised and analyzed by amino acid analyzer for arginine.

As shown in FIG. 11, AAD fusion protein (SEQ ID NO: 40, FIG. 3E), even at the lowest dosage of 100 μg, depletes plasma arginine efficiently at Day 1, 3 and 5, suggesting that AAD can deplete arginine in vivo efficiently for at least 5 days. The arginine level returns to normal gradually at Day 6 and Day 7 in all treatment groups.

Example 7

In Vivo Efficacy of AAD Fusion Protein on Cancer Cell Xenografts

Nude balb/c mice (5-7 weeks) are used in this study and they are allowed to acclimatize for a week before the experiment. Mice are inoculated subcutaneously with 2×10⁶ cancer cells in 100 μl of fresh culture medium. Ten days later, the mice are randomly separated into control and treatment group. Control group receives 100 μl PBS and treatment group receives 100 μl AAD fusion protein intraperitoneally weekly. Tumor size is measured by caliper and tumor volume is calculated using formula: (length×width²)/2. Blood draw are obtained at Day 5 after each treatment for plasma measurement of arginine.

Claims

1. An albumin-binding arginine deiminase fusion protein comprising a first portion comprising one or two components selected from an albumin-binding domain, an albumin-binding peptide or an albumin-binding protein(s) fused to a second portion comprising arginine deiminase to form the albumin-binding arginine deiminase fusion protein, and one or more linker molecules; the first portion being positioned far from active site of the second portion by said linker molecule such that the albumin-binding arginine deiminase fusion protein retains the activity of arginine deiminase and binds serum albumin with neither function of one portion of the fusion protein being interfered with by the other portion of the fusion protein, wherein pegylation of said arginine deiminase is avoided, and wherein the albumin-binding arginine deiminase fusion protein comprises a sequence selected from seq id NO: 36, 37, 38, 39, 40, or 41.

2. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the two components of the first portion are the same.

3. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the two components of the first portion are different.

4. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the albumin-binding domain is seq id NO: 46, 47, 48, or 49.

5. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the albumin binding peptide is seq id NO: 46, 47, 48, or 49.

6. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the albumin binding protein is seq id NO: 46, 47, 48, or 49.

7. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the linker molecule comprises a sequence selected from seq id NO: 50, 51, 52, 53, or serine-glycine-serine (SGS) amino acid sequence.

8. The albumin-binding arginine deiminase fusion protein of claim 1 further comprising at least one of Poly-N or a His tag.

9. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the fusion comprises a remaining portion of an intein-mediated protein ligation between the first portion and the second portion.

10. The albumin-binding arginine deiminase fusion protein of claim 9 wherein the intein-mediated protein comprises a chitin binding domain.

11. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the arginine deiminase is selected from arginine deiminase produced from a Mycoplasma, Lactococcus, Pseudomonas, Streptococcus, Escherichia, Mycobacterium or Bacillus microorganism.

12. The albumin-binding arginine deiminase fusion protein of claim 11 wherein the arginine deiminase is produced from Mycoplasma arginini, Lactococcus lactis, Bacillus licheniformis, Bacillus cereus, Mycoplasma arthritidis, Mycoplasma hominis, Streptococcus pyogenes, Streptococcus pneumoniae, Mycobacterium tuberculosis, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas aeruginosa or a combination thereof.

13. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the fusion protein is formed by reacting the arginine deiminase having a N-terminal cysteine residue with a reactive thioester at C-terminus of the albumin-binding domain so that the arginine deiminase and the albumin-binding domain are linked by a covalent bond.

14. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the fusion protein is formed by reacting the albumin-binding domain having a N-terminal cysteine residue with a reactive thioester at C-terminus of the arginine deiminase so that the arginine deiminase and the albumin-binding domain are linked by a covalent bond.

15. The albumin-binding arginine deiminase fusion protein of claim 1 wherein the fusion protein is formed by using seq id NO: 42 and 43 and by reacting the arginine deiminase having a N-terminal cysteine residue with a reactive thioester at C-terminus of the albumin-binding domain so that the arginine deiminase and the albumin-binding domain are linked by a covalent bond.

16. A pharmaceutical composition comprising the albumin-binding arginine deiminase fusion protein of claim 1 in a pharmaceutically-acceptable carrier.

17. The pharmaceutical composition of claim 16 wherein the composition has a pH in a range of 5.5 to 9.5.

18. The pharmaceutical composition of claim 16 wherein the composition has a pH of 7.4.

19. The pharmaceutical composition of claim 16 wherein the composition has a pH of 6.5.