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EP-4737471-A1 - METHODS FOR THE C-TERMINAL MODIFICATION OF (POLY)PEPTIDES

EP4737471A1EP 4737471 A1EP4737471 A1EP 4737471A1EP-4737471-A1

Abstract

The present invention concerns a method for the C-terminal modification of a (poly)peptide that is free of cysteine residues comprising providing the to-be-modified (poly)peptide as a fusion protein with a C-terminal tag of the amino acid sequence C(X) n , wherein n is an integer of 1 to 100, and X is any amino acid with the exception of C, reacting said fusion protein with 2-nitro-5-thiocyanatobenzoic acid or salt thereof under conditions that allow to cyanylate the cysteine residue of the C-terminal tag, and contacting the cyanylated fusion protein with a nucleophile selected from the group consisting of ammonia, an ammonium salt or a primary amino group containing agent under conditions that allow cleaving the cyanylated fusion protein and obtain the C-terminally modified (poly)peptide of interest. The invention also encompasses artificial (poly)peptides that comprise a (poly)peptide of interest and the amino acid sequence C(X) n fused to its C-terminus.

Inventors

  • SCHWARZ, CHRISTIAN
  • REBMANN, Philipp
  • STRAUSS, Jannik
  • SEIDE, Selina

Assignees

  • NUMAFERM GmbH

Dates

Publication Date
20260506
Application Date
20241029

Claims (15)

  1. Method for the C-terminal modification of a (poly)peptide of interest, wherein said (poly)peptide of interest is free of cysteine residues and at least 10 amino acids in length, said method comprising: a) providing a fusion protein comprising the (poly)peptide of interest with the amino acid sequence C(X) n fused to its C-terminus, wherein n is an integer of 1 to 100, and X is any amino acid with the exception of C, wherein the fusion protein is an artificial, non-natural protein or fragment thereof in that the C-terminal amino acid sequence C(X) n does not naturally occur in combination with the (poly)peptide of interest; b) reacting the fusion protein with 2-nitro-5-thiocyanatobenzoic acid (NTCB) or salt thereof to cyanylate the cysteine residue; c) contacting the cyanylated fusion protein with a nucleophile selected from the group consisting of ammonia, an ammonium salt or a primary amino group containing agent to cleave the cyanylated fusion protein and obtain the C-terminally modified (poly)peptide of interest.
  2. The method of claim 1, wherein (1) steps b) and c) are carried out at a pH of 8.0-10.0, preferably at pH 9.0; and/or (2) steps b) and c) are carried out simultaneously, preferably by adding NTCB and the nucleophile at the same time.
  3. The method of claim 1 or 2, wherein in step c) ammonia or ammonium hydroxide is used as the nucleophile and the C-terminal modification is an amidation.
  4. The method of any one of claims 1 to 3, wherein (1) n is 1 to 50, preferably 1 to 20 or 1 to 15 or 1 to 10 or 1 to 5 or 1 to 2 or n is 1; and/or (2) n is selected such that the amino acid sequence C(X) n has a length of at least 5% of the total length of the (poly)peptide of interest.
  5. The method of claim 1 or 2, wherein in step c) a monomeric amino acid or a peptide of 2 to 100 amino acids, preferably 2 to 50 or 2 to 25 or 2 to 15 amino acids, in length is used as the amino group containing agent and the C-terminal modification is a C-terminal elongation by said amino acid or peptide, wherein said amino acid is not C, and preferably not G, or said peptide does not contain a C residue.
  6. The method of claim 5, wherein n is 1 or n is selected such that the C-terminal tag consisting of the amino acid sequence C(X) n differ in length by at least 1, preferably by at least 2 amino acids.
  7. The method of claim 6, wherein said difference in length is at least 5% of the total length of the (poly)peptide of interest.
  8. The method of any one of claims 1 to 7, wherein the (poly)peptide of interest is 10 to 1000 amino acids in length, preferably 10 to 500 amino acids or 15 to 250 amino acids or 20 to 150 amino acids or 20 to 100 amino acids or 20 to 50 amino acids.
  9. The method of any one of claims 1 to 8, wherein the method further comprises the step of purifying or enriching the C-terminally modified (poly)peptide of interest, preferably relative to the non-modified (poly)peptide of interest, wherein purification or enrichment is optionally carried out by chromatography, preferably size exclusion chromatography, affinity chromatography or HPLC.
  10. The method of any one of claims 1 to 9, wherein the fusion protein and the C-terminally modified (poly)peptide of interest obtained in step c) differ in length by at least 1 amino acid, preferably by at least 2 amino acids, preferably the fusion protein differs from the C-terminally modified (poly)peptide of interest in molecular mass by at least 2.5%, preferably by at least 5%.
  11. The method of any one of claims 1 to 10, wherein the fusion protein is recombinantly produced in a host organism.
  12. The method of any one of claims 1 to 11, wherein the fusion protein further comprises an amino acid sequence derived from an RTX protein, preferably derived from HlyA, that preferably facilitates renaturation of the (poly)peptide of interest fused to the N-terminus of the (poly)peptide of interest.
  13. The method of any one of claims 1 to 12, wherein the amino acid sequence (X) n is X 1 (X) n-1 , wherein (1) X 1 is not W, preferably X 1 is not W, Y, F, or P; and/or (2) X 1 is an unpolar amino acid, preferably A, V, L or I, more preferably L, a basic amino acid, preferably K or R, more preferably K, or an acidic amino acid, preferably D or E.
  14. The method of any one of claims 1 to 13, wherein the (poly)peptide of interest has biological activity, preferably in a mammal, more preferably in a human, or is useful as a pharmaceutical for a mammal, preferably a human.
  15. Isolated (poly)peptide comprising a (poly)peptide of interest and the amino acid sequence C(X) n fused to its C-terminus, wherein n is an integer of 1 to 100, and X is any amino acid with the exception of C, wherein said (poly)peptide is an artificial (poly)peptide in that the (poly)peptide of interest and said amino acid sequence C(X) n are artificially combined, and wherein the isolated (poly)peptide optionally further comprises an amino acid sequence derived from an RTX protein, preferably derived from HlyA, that facilitates or increases renaturation of the (poly)peptide of interest fused to the N-terminus of the (poly)peptide of interest.

Description

FIELD OF THE INVENTION The present invention is in the field of molecular biology and (poly)peptide synthesis and relates to methods for the C-terminal modification of a (poly)peptide including cyanylation with 2-nitro-5-thiocyanatobenzoic acid or salt thereof and subsequent cleavage using a nucleophile. BACKGROUND OF THE INVENTION To date, recombinant protein/enzyme production for use in industrial processes is widely established. It is expected that in the future more and more industrial processes that are currently based on traditional chemistry will be adapted to involve recombinant technologies. Recent years have seen an increasing demand for (poly)peptides and proteins for various purposes, including pharmaceuticals. Many of these peptides, polypeptides, and proteins are produced recombinantly by expression in a host organism and subsequent isolation. While said production method provides for many advantages, it is notoriously difficult to produce peptides that are modified in that they have non-naturally occurring modifications or modifications that cannot be made by the producing host organism. These modifications include C-terminal modifications, such as amidation or the addition of certain tags. While such modifications may be added by chemical synthesis, many of the (poly)peptides and proteins are susceptible to degradation or denaturation under the reaction conditions required for such modification. There is thus need in the art for the provision of methods that allow the easy modification of (poly)peptides and proteins without adversely affecting their stability, integrity or three-dimensional conformation. The present methods solve this need by providing a method for the simply and efficient C-terminal modification of (poly)peptides and proteins with a wide range of nucleophilic agents while allowing easy separation and purification of the obtained modified (poly)peptides. SUMMARY OF THE INVENTION In a first aspect, the present invention concerns a method for the C-terminal modification of a (poly)peptide of interest, wherein said (poly)peptide of interest is free of cysteine residues and at least 2 amino acids in length, said method comprising: a) providing a fusion protein comprising the (poly)peptide of interest with the amino acid sequence C(X)n fused to its C-terminus, wherein n is an integer of 1 to 100, and X is any amino acid with the exception of C, and wherein preferably the fusion protein is an artificial protein;b) reacting the fusion protein with 2-nitro-5-thiocyanatobenzoic acid (NTCB) or salt thereof to cyanylate the cysteine residue; andc) contacting the cyanylated fusion protein with a nucleophile selected from the group consisting of ammonia, an ammonium salt or a primary amino group containing agent to cleave the cyanylated fusion protein and obtain the C-terminally modified (poly)peptide of interest. In various embodiments of these methods steps b) and/or c), preferably both, are carried out at a pH of 8.0-10.0, preferably at pH 9.0. In various embodiments, steps b) and c) are carried out simultaneously, preferably by adding NTCB and the nucleophile at the same time. Alternatively, steps b) and c) can be carried out subsequently. In step c) ammonia or an ammonium salt, such as ammonium hydroxide or ammonium acetate, may be used, typically in form of an aqueous solution or an ammonia-containing buffer. In such embodiments, the C-terminal modification is an amidation of the terminal carboxy group. According to the invention, n is at least 1, preferably n is 1 to 20 or 1 to 17 or 1 to 12 or 1 to 10 or 1 to 6 or 1 to 2 or n is 1; and/or n is selected such that the amino acid sequence C(X)n has a length that is at least 5% of the total length of the (poly)peptide of interest. In various other embodiments, in step c) a monomeric amino acid or a peptide of 2 to 100 amino acids, preferably 2 to 50 or 2 to 25 or 2 to 15 amino acids, in length is used as the nucleophile, in particular as the amino group containing agent. In such embodiments, the C-terminal modification is a C-terminal elongation by said amino acid or peptide. In various embodiments, said amino acid is not C or said peptide used as the nucleophile does not contain a C residue, in particular in instances where steps b) and c) are carried out simultaneously. In various embodiments, said monomeric amino acid is not G. In such embodiments, n may be 1 or n is selected such that the C-terminal tag consisting of the amino acid sequence C(X)n differ in length by at least 1, preferably by at least 2 amino acids. In various embodiments, said difference in length is at least 5% of the total length of the (poly)peptide of interest. In various embodiments of the methods disclosed herein, the (poly)peptide of interest is 4 or 5 to 1000 amino acids in length, preferably 5 or 10 to 500 amino acids or 15 to 250 amino acids or 20 to 150 amino acids or 5 to 100 amino acids or 4 to 50 amino acids. In various embodiments, the method may f