MCP-1 analogs

Details MCP-1 analogs MCP-1 analogs

2000-01-27

2002-05-07

Kemmerer, Elizabeth (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S320100, C435S325000, C514S002600, C530S300000, C424S085100

Reexamination Certificate

active

06383782

ABSTRACT:

The present invention relates to novel analogues of Monocyte Chemoattractant Protein-1 (MCP-1) and corresponding potynucleotide sequences vectors. host cells and recombinant expressions particularly in
E. coli.
MCP-1 is a member of the chemokine family of pro-inflammatory cytokines which mediate leukocyte chemotaxis and activation. MCP-1 is a C—C chemokine which is one of the most potent and selective T-cell and monocyte chemoattractant and activating agents known. MCP-1 has been implicated in the pathophysiology of a large number of inflammatory diseases including rheumatoid arthritis (RA), glomerular nephritides, lung fibrosis, restenosis (International Patent Application WO 94/09128), alveolitis (Jones et al. 1992, J. Immunol. 49, 2147) and asthma. Other disease areas where MCP-1 is thought to play a part in their pathology are: atherosclerosis (e.g. Koch et al. 1992 J. Clin. Invest. 90, 772-779); psoriasis (Deleuran et al. 1996 J. Dermatological Science 13, 228-236), delayed -type hypersensitivity reactions of the skin; inflammatory bowel disease (Grimm et al. 1996 J. Leukocyte Biol. 59, 804-812), multiple sclerosis and; brain trauma (Berman et al. 1996 J. Immunol. 156, 3017-3023). An MCP-1 inhibitor may also be useful to treat stroke, reperfusion injury, ischemia, myocardial infarction and transplant rejection.
It is known that analogues of MCP-1 can be prepared which are antagonistic to its biological properties. Rollins in U.S. Pat. No. 5,459,128 describes MCP-1 antagonists which include truncation at the N-terminus of the protein. Gong in J. Exp. Med. (1995) 181, 631, describes a particular MCP-1 antagonist of this type which is MCP1(9-76) (SEQ ID NO: 30). Gong made MCP-1(9-76) (SEQ ID NO: 30) by direct chemical synthesis. Chemical synthesis is not a commercially attractive process for manufacture of MCP-1(9-76) (SEQ ID NO: 30). Lewis & Gong in Canadian patent application 2152141 describe chemically synthesised MCP-1 analogues which are truncated at the N-tenninus. Conservative amino acid substitutions for some amino acids (excluding valine) are also described therein (see page 7) but preferably only at regions beyond position 35 of MCP-1.
A recombinant method would be preferable for large scale commercial manufacture of MCP1(9-76) (SEQ ID NO: 30) but to date there has been no specific experimental disclosure of any such recombinant methodology.
A preferred organism for recombinant manufacture is
E. coli
because of its ease of handling. However, Rollins U.S. Pat. No. 5,459,128 warns of problems with use of bacterial expression systems due to incorrect protein folding and states a preference for COS cell expression systems (see column 2, lines 49-51). However, COS cells are not desirable for large scale manufacture because they only provide a transient expression system. We have discovered in our experiments (which are presently unpublished) that secretory expression (i.e. using a leader sequence to direct expressed protein through the cytoplasmic membrane) of MCP1(9-76) (SEQ ID NO: 30) in
E. coli
has the disadvantage of giving poor yields (see Comparative Example 2). Furthermore we have also discovered that when a polynucleotide encoding MCP1(9-76) is expressed intracellularly in
E. coli
the resulting product is undesirably heterogeneous due to the presence of an additional methionine residue at the N-terminus in a significant proportion (typically 50%) of the product. There thus exists a need for an improved way of making a MCP1(9-76) (SEQ ID NO: 30) type protein, particularly in
E. coli.
The present invention is based on the discovery that substitution of an alanine, glycine or threonine for the natural valine in position 9 of MCP1(9-76) gives a novel MCP-1 analogue (termed “[V9A]MCP1(9-76)” (SEQ ID NO: 9), “[V9G]MCP1(9-76)” (SEQ ID NO: 26) or “[V9T]MCP1(9-76)” (SEQ ID NO: 29) respectively herein) which can be recombinantly manufactured in
E. coli
to produce a substantially homogeneous product (at least substantially lacking unwanted methionine at the N-terminus) in good yield which retains the antagonistic effect of MCP1(9-76) as measured in the chemotaxis assay described herein.
According to one aspect of the present invention there is provided a protein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29). The protein sequences of [V9A]MCP1(9-76), [V9G]MCP1(9-76) and [V9T]MCP1(9-76) are set out in SEQ ID NO: 9, 26 and 29 respectively. Preferably the protein is selected from [V9A]MCP1(9-76) (SEQ ID NO: 9) or [V9G]MCP1(9-76) (SEQ ID NO: 26) and of these [V9A]MCP1(9-76) (SEQ ID NO: 9) is especially preferred.
Note Lewis & Gong in Canadian patent application 2152141 describe conservative amino acid substitutions in truncated MCP-1 analogues but these are preferably beyond position 35 (of MCP-1) and furthermore that disclosure is completely silent on substitution of valine anywhere in the molecule. Preferably the [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29) is essentially free of methionine at its N-terminus. The term “essentially free” in this context means that methionine is present at a level of about 10% or less (when) analysed by any one of reverse phase HPLC, capillary zone electrophoresis, Edman degradation and electrospray mass spectrometry) at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29), preferably methionine is present at a level of less than 5% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) or [V9T]MCP1(9-76) (SEQ ID NO: 29), preferably methionine is present at a level of less than 3% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9-76) (SEQ ID NO: 29), more preferably methionine is present at a level of less than 1% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29), more preferably methionine is present at a level of less than 0.3% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29) and more preferably methionine is present at a level of less than 0.1% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29). Preferably the [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26). [V9T]MCP1 (9-76) (SEQ ID NO: 29) gives a single peak when analysed by reverse phase HPLC.
According to another aspect of the invention there is provided a polynucleotide sequence encoding a protein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) OR [V9T]MCP1(9-76) (SEQ ID NO: 29). The polynucleotides of the present invention may be in the form of RNA or in the form of DNA including synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to any coding sequence shown herein or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptides. The polynucleotides which encode the mature polypeptide may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader sequence; and optionally non-coding sequence 5′ and/or 3′ of the coding sequence for the mature polypeptide. Thus, the term “polynucleotide sequence encoding a protein selected from [V9A&

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