Porcine G-CSF variants and their uses

Abstract

The present invention relates to variants of porcine granulocyte colony stimulating factor (pG-CSF). The pG-CSF variants are useful in treating preventing or reducing the incidence of bacterial infections in swine. Methods of treating swine are disclosed.

Claims

1. A porcine granulocyte colony stimulating factor (pG-CSF) variant consisting of a sequence of: X.sub.1PLSPASSLPQSFLLKX.sub.2LEQVRKIQADGAELQERLCATHKLC(pAF)PQELVLLGHSLGL PQASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPELAPALDILQLDVTDLATN IWLQX.sub.3EDLRX.sub.3APASLPTQGTVPTFTSAFQRRAGGVLVVSQLQSFLELAYRVLRYLAEP (SEQ ID NO: 13); wherein X.sub.1is selected from the group of methionine alanine, norleucine alanine, alanine only, and no amino acids; wherein X.sub.2 is cysteine or serine; wherein X.sub.3 is methionine or norleucine; and wherein a para-acetyl phenylalanine (pAF) synthetic amino acid present at position 43 is covalently attached to a poly(ethylene glycol) (PEG).

2. The pG-CSF variant of claim 1, wherein the PEG has a molecular weight of about 20 kD to about 50 kD.

3. The pG-CSF variant of claim 1, wherein the PEG has a molecular weight of about 30 kD.

4. The pG-CSF variant of claim 1, wherein the PEG is linear.

5. The pG-CSF variant of claim 1, wherein a para-acetyl phenylalanine (pAF) synthetic amino acid present at position 43 is covalently attached to a 30 kD linear PEG.

6. A pharmaceutical composition comprising the pG-CSF variant of claim 1, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

7. A method for treating MMA syndrome in a porcine comprising administering a therapeutically effective amount of the pGCSF variant of claim 1 to the porcine in need thereof.

8. The method of claim 7, wherein the MMA syndrome comprises symptoms of mastitis, metritis and/or agalactia.

9. The method of claim 7, wherein the porcine is a periparturient sow.

10. The method of claim 7, wherein the therapeutically effective amount of pG-CSF is about 10-100 μg/kg animal weight.

11. The method of claim 7, wherein the therapeutically effective amount of pG-CSF is about 30-50 μg/kg animal weight.

12. The method of claim 7, wherein the administering occurs at least once within 7 days prior to farrowing.

13. The method of claim 7, wherein the administering occurs at farrowing.

14. The method of claim 12, further comprising a second administration no later than 14 days after farrowing.

15. A method for increasing blood neutrophils in a porcine comprising administering a therapeutically effective amount of the pG-CSF variant of claim 1 to the porcine.

16. The method of claim 15, wherein the therapeutically effective amount of pGCSF is about 10-100 μg/kg animal weight.

17. The method of claim 15, wherein the administering occurs at least once within 7 days prior to farrowing.

18. The method of claim 15, wherein the administering occurs at farrowing.

19. The method of claim 17, further comprising a second administration no later than 14 days after farrowing.

20. A method for stimulating innate immune response by increasing blood neutrophils in a porcine comprising administering a therapeutically effective amount of the pG-CSF variant of claim 1 to the porcine in need thereof.

Description

EXAMPLE 1

(1) Different variants of the porcine granulocyte colony stimulating factor (pG-CSF) cDNA (GenBank Accession number U68481.1) are generated by introducing a TAG stop codon in the selected positions by site-directed PCR mutagenesis. Also, the portion of the cDNA encoding the signal sequence is replaced by a single methionine codon (i.e. ATG). For example, a cDNA encoding wild type mature pG-CSF with the altered signal sequence could be: 1 atggcccctc tcagccctgc cagctccctg ccccagagct tcctgctcaa gtgcttagag 61 caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg tgccacccac 121 aagctgtgcc acccccagga gctggtgctg ctcgggcact ctctgggcct cccccaggct 181 tccctgagca gctgctccag ccaggccctg cagctgactg gctgcctgaa ccaactgcat 241 ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc cccagagctg 301 gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa catctggctg 361 cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac cgtgccgacc 421 ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca gctgcagagc 481 ttcctggagc tggcgtaccg tgtcctgcgc tacctcgccg agccctga
(SEQ ID NO: 5). This variant would encode a polypeptide of: 1 MAPLSPASSL PQSFLLKCLE QVRKIQADGA ELQERLCATH KLCHPQELVL LGHSLGLPQA 61 SLSSCSSQAL QLTGCLNQLH GGLVLYQGLL QALAGISPEL APALDILQLD VTDLATNIWL 121 QMEDLRMAPA SLPTQGTVPT FTSAFQRRAG GVLVVSQLQS FLELAYRVLR YLAEP
(SEQ ID NO: 1). If the TAG stop codon is desired to replace the H43 residue, the cDNA sequence could be: 1 atggcccctc tcagccctgc cagctccctg ccccagagct tcctgctcaa gtgcttagag 61 caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg tgccacccac 121 aagctgtgct agccccagga gctggtgctg ctcgggcact ctctgggcct cccccaggct 181 tccctgagca gctgctccag ccaggccctg cagctgactg gctgcctgaa ccaactgcat 241 ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc cccagagctg 301 gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa catctggctg 361 cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac cgtgccgacc 421 ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca gctgcagagc 481 ttcctggagc tggcgtaccg tgtcctgcgc tacctcgccg agccctga
(SEQ ID NO: 6). The resulting polypeptide would be: 1 MAPLSPASSL PQSFLLKCLE QVRKIQADGA ELQERLCATH KLCxPQELVL LGHSLGLPQA 61 SLSSCSSQAL QLTGCLNQLH GGLVLYQGLL QALAGISPEL APALDILQLD VTDLATNIWL 121 QMEDLRMAPA SLPTQGTVPT FTSAFQRRAG GVLVVSQLQS FLELAYRVLR YLAEP
(SEQ ID NO: 2), where the “x” indicates the H43 residue replaced with a synthetic amino acid, as described below.

(2) Plasmids encoding the variants are transformed into E. coli cells containing the expanded genetic code system components for incorporation of the synthetic amino acid para-acetyl phenylalanine (pAF). Transformed cells are grown in media supplemented with pAF and induced to express pG-CSF with pAF incorporated into the sites indicated. The expression system has been described, for example, in WO 2010/011735 (incorporated herein by reference), and is generally known in the art.

(3) Expression of the transfected cDNA variants is induced with arabinose, the cells are harvested, and the target pG-CSF pAF site variants are isolated and purified by reverse phase high-pressure liquid chromatography (RP-HPLC). An activated 30 kD linear aminooxy-PEG is site-specifically conjugated to the incorporated pAF. PEG-pG-CSF conjugates are purified from excess PEG and unconjugated pG-CSF variants by chromatography.

EXAMPLE 2

(4) The in vitro biological activity of PEGylated pG-CSF (PEG-pG-CSF) variants is measured by the ability of the variants to induce proliferation of M-NSF-60 cells (ATCC CRL-1838). The concentration of the variants able to effect 50% of maximal proliferation (EC.sub.50) is determined by comparison to a standard curve generated with wild type (WT) pG-CSF. Based on the results of expression as presented in Example 1 and the biological assays described here, PEG-pG-CSF variants are selected for further study.

(5) The in vivo activity of the selected candidate PEG-pG-CSF variants is tested in a rodent model. Sprague Dawley rats (3/group) are treated with 0.25 mg/kg body weight with a PEG-pG-CSF variant. Blood samples are taken at 0 (pre-dosing), 1, 3, 6, 24, 48, 56, 72, 96, 144, 192, and 264 hours for pharmokinetic (PK) analysis, and samples are taken at 24, 48, 72, and 96 hours for a complete blood count (CBC analysis). The primary measurement in the CBC analysis is the number of neutrophils present. A H43 variant stimulates a higher level of neutrophil development than other variants tested. All variants have similar PK profiles.

EXAMPLE 3

(6) The PEG-pG-CSF H43pAF is prepared as follows. As in Example 1, expression of the transfected cDNA variants is induced with arabinose, the cells are harvested, and the pG-CSF H43pAF site variant is isolated, denatured and refolded, and purified by cation exchange liquid chromatography (CEX), using CAPTO Adhere Impres (GE Healthcare Lifesciences). Briefly, the unpegylated pG-CSF H43pAF variant is loaded onto the column to a concentration of 1-5 mg/mL resin. The column is washed with five column volumes (CV) 30 mM sodium acetate at pH 4.5. Elution of the pG-CSF H43pAF variant is with a linear gradient of elution buffer (30 mM sodium acetate, 0.5 M NaCl, Ph 4.5), by washing with 0-100% elution buffer over 20 CV.

(7) Based on mass spectroscopy (MS) analysis of pG-CSF H43pAF, the isolated peptides include a main peak represented by SEQ ID NO: 2 and several different contaminants. The contaminants include loss of the N-terminal methione (SEQ ID NO: 4), loss of both the N-terminal methionine and alanine (SEQ ID NO: 8), and substitution of norleucine for the N-terminal methionine (SEQ ID NO: 7). Norleucine is known to be misincorporated instead of the amino acid methionine in high density fermentation with E. coli. Norleucine incorporation is reduced by using one or more of the following steps: feeding the fermentation solutions with methionine; fermenting with complex media instead of defined media (the complex media has one or more non-defined components in it including but limited to glycerol, salts, amino acids, vitamins, yeast extracts, plant and animal hydrolysates, peptones, and tryptones); and/or lowering the temperature of the fermentation reaction mixture post induction.

(8) The pG-CSF H43pAF variant is taken from the cation exchange chromatography pool after using Capto SP Impres chromatography and buffer exchanged into 30 mM sodium acetate, 4% sucrose, pH 4.0 using a 10 kDa MWCO tangential flow filtration cassette. The pG-CSF H43pAF variant is then concentrated to about 8.0 mg/mL using an Amicon Ultra centrifugal filter according to manufacturer's instructions. Once concentrated, 30K linear PEG (PEG can be purchased commercially from NOF America Corporation or EMD Merck, for example) is added in a 6:1 molar ratio of PEG to pG-CSF H43pAF variant. The PEG/pG-CSF variant mixture is then incubated at about 28° C. for at least 21 hours. This method results in >98% of the pG-CSF variant being conjugated with PEG. The pegylated variant can then be purified by CEX as above. When tested in the M-NSF-60 cell bioassay (Example 2), the PEG-pG-CSF H43pAF variant has an EC.sub.50 of at least 0.40 ng/mL, demonstrating good binding and potency characteristics.

(9) Samples are frozen and thawed over five cycles by freezing at 0° C. in 1.5 mL tubes and thawing in a room temperature water bath. No significant impact is observed for the high molecular weight (HMW) protein profile over five cycles of freeze-thawing, demonstrating the stability of the variant in solution.

(10) Two additional pG-CSF H43pAF variants are generated to attempt to improve refolding efficiency and thermostability of the variant at 50° C. Cysteine 17 is changed to either alanine (C17A) or to serine (C17S, SEQ ID NOs: 9-12). These mutations do not improve refolding yield, but C17A has decreased thermostability. PEG-pG-CSF H43pAF/C17S does have a slightly improved EC.sub.50 of 0.26 ng/mL.

EXAMPLE 4

(11) The PEG-pG-CSF H43pAF variant is administered to sows to characterize changes in blood neutrophils. Six sows of 1.5-5 years of age and an average body weight of 269.7 kg are given 40 μg/kg of the PEG-pG-CSF H43pAF variant by intramuscular injection on the side of the neck. The PEG-pG-CSF H43pAF variant is suspended in 30 mM sodium citrate, 250 mM arginine, pH 6.0 at a concentration of 8.2 mg/mL. Animals do not receive any concomitant medication following initiation of treatment. No adverse events are observed.

(12) Blood is taken on day 0 prior to dosing and on days 2, 7, 10, 14, 17 and 21 post-dosing. Neutrophil counts are determined for each sow and a mean for the treatment is determined for each day. Treatment with a single dose of the PEG-pG-CSF H43pAF variant results in measurable increases in blood neutrophil counts over a three-week period (Table 1). Additional doses would be expected to stimulate maintenance of the higher neutrophil levels.

(13) TABLE-US-00001 TABLE 1 Effect of PEG-pG-CSF H43pAF variant on mean daily blood neutrophil counts. Day 0 2 7 10 14 17 21 Neutrophils (1000/μL) 4.65 43.68 25.08 30.1 18.53 15.14 9.83

EXAMPLE 5

(14) The PEG-pG-CSF H43pAF variant is administered to periparturient sows to characterize the effect on mastitis, metritis, and agalactia (MMA) syndrome and on piglet survival.

(15) Sows at 95-100 days of gestation are placed into farrowing crates and hygienic husbandry practices are followed until day 107 of gestation for each sow. On day 107, blood is collected and then the sows (25 per group) are treated either with 40 μg/kg of the PEG-pG-CSF H43pAF variant as in Example 4 or with a sodium chloride solution as a negative control. The sows are then placed in unhygienic conditions to stimulate development of MMA. The unhygienic conditions include placing a mat on the grated floor of the farrowing crate to allow bedding and waste material to accumulate. Also, a mixture of water, feces, and pine sawdust (2:1:1) is used to contaminate the crates. No oxytocic or corticosteroid drugs are given to the sows. Clinical observations and rectal temperatures are collected on each sow once daily in the morning beginning on day 107 of gestation and continuing until a diagnosis of MMA at which time the sow has all samples collected and is then removed from the study, given treatment, and has hygienic crate conditions returned. Samples collected include blood, rectal temperature, and swabs of infected glands.

(16) Farrowing typically occurs on day 114 of gestation. Piglets are weighed and tagged within 12 hours of birth. Clinical observations of the piglets are made twice daily, and weights are also measured on days 3, 7, and 21 after birth.

(17) Using the per protocol definition of disease, little difference was observed between the two groups. However, when vulvar discharge was removed from the definition of disease, the incidence of disease was reduced by more than 50% in the PEG-pG-CSF treated group. The treated group also had a greater number of piglets weaned compared to control group (Table 2).

(18) TABLE-US-00002 TABLE 2 Effect of PEG-pG-CSF on MMA incidence and piglet survival. Variable Control PEG-pG-CSF P value Sows (number/group) MMA 36% (9/25) 32% (8/35) 1.0000 MMA excluding vulvar 28% (7/25) 12% (3/25) 0.2890 discharge Piglets (Std. Error of Mean) Number born/litter 13.3 (0.78) 13.1 (0.72) 0.8567 Number weaned/litter 8.3 (0.61) 9.4 (0.61) 0.2056 Percent survival 65.3 (4.5) 72.4 (4.3) 0.2590 Range survival/litter 0-100% 38-100% n/a Weaning weight 5.9 (0.21) 5.7 (0.19) 0.5944