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CN-122029276-A - Macrocyclic peptide libraries displayed on the surface of yeast cells

CN122029276ACN 122029276 ACN122029276 ACN 122029276ACN-122029276-A

Abstract

The present invention relates to the generation of libraries of cysteine-enriched macrocyclic peptide sequences expressed on the surface of yeast cells, methods of their preparation, and yeast cell populations genetically modified to express a plurality of disulfide-tethered macrocyclic peptide ligands having different structures and amino acid sequences on the surface of yeast cells.

Inventors

  • A. ANGELINI
  • S. Lin Qiano
  • Y. Mazokato
  • Z. Roman Nuck

Assignees

  • 阿尔扎尼亚有限责任公司

Dates

Publication Date
20260512
Application Date
20241016
Priority Date
20231020

Claims (11)

  1. 1. A yeast cell comprising an expression vector encoding a fusion protein (I): (NH 2 -or COOH) - [ MP ] - [ FL ] - [ IFT ] - [ CSP ] - (COOH or NH 2 ) (I) Wherein: (i) (NH 2 or COOH) represents the amino or carboxyl terminus of the subject cysteine-enriched macrocyclic peptide [ MP ] sequence, and (COOH or NH 2 ) represents the corresponding carboxyl or amino terminus of the protein [ CSP ] expressed on the surface of yeast cells, wherein the first terminus is extracellular; (ii) [ MP ] represents a macrocyclic peptide (X) n C(X) m C(X) t sequence in which two cysteine residues "C" form a disulfide bond, n, m, t are 0 or integers, wherein n=0-5;m =3-21, t=0-5, provided that n+m+t=3-21; Or (b) [ MP ] represents a macrocyclic peptide (X) n C(X) m C(X) t C(X) y sequence in which two cysteine residues "C" form a disulfide bond, n, m, t and y are 0 or integers, wherein n=0-5;m =0-22, t=0-22, y=0-5, provided that n+m+t+y=4-22; Or (b) [ MP ] represents a macrocyclic peptide (X) n C(X) m C(X) t C(X) j C(X) y in which each cysteine residue "C" forms a disulfide bond, n, m, t, j, y is 0 or an integer, wherein n=0-5, m=0-21, t=0-21, j=0-21, y=0-5, provided that n+m+t+j+y=4-21; "X" means any natural (cAA) or unnatural (NcAA) amino acid, where: -the natural amino acid (cAA) is encoded by any of 61 naturally occurring codons, or it is a random amino acid encoded by a degenerate codon selected from NNN, NNK, NNS, NDT, DBK, NRT, NNC, NWW, RVK; DVT, NVT, VVC, RST, NTT, TDK, provided that there is at least one random amino acid in the sequence of the macrocyclic peptide of interest, preferably 20 to 50% of the amino acids in the sequence are random, more preferably >50% of the amino acids are random; -the unnatural amino acid (NcAA) is encoded by a TAG, TAA or TGA stop codon present within the coding sequence of the peptide of interest in the expression vector; (iii) [ FL ] represents a spacer sequence containing 1 to 25 amino acids, preferably selected from the group consisting of (GGGGS) n , wherein n ranges from 1 to 5, (G) n , wherein n is an integer from 1 to 25, (S) n , wherein n is an integer from 1 to 25, (P) n , wherein n is an integer from 1 to 25, (EAAAK) n , wherein n is an integer from 1 to 5, (A (EAAAK) 4 ALEA(EAAAK) 4 A) n , wherein n is an integer from 1 to 2, (XP) n , wherein n is an integer from 1 to 12, and "X" may be any natural amino acid, preferably alanine (A), lysine (K) and glutamic acid (E); (iv) [ IFT ] represents a sequence suitable for detecting and/or isolating a yeast cell expressing MP-CSP or CSP-MP fusion on the cell surface; (v) [ CSP ] represents a protein expressed on the surface of a yeast cell, which can be fused to the N-terminus or C-terminus of a macrocyclic peptide [ MP ] sequence, comprising: -at least one extracellular portion; a transmembrane moiety, or a moiety that binds to a membrane and/or wall, the moiety being covalently or non-covalently anchored to a molecule of the membrane and/or wall.
  2. 2. The yeast cell of claim 1, wherein the [ CSP ] is an Aga1 protein, an Aga2 protein, a Glycosyl Phosphatidyl Inositol (GPI) binding protein family member, preferably Cwp1p, cwp2p, tip1p, flop1p, sed1p, YCR89, tir1p, or a synthetic, non-naturally occurring protein capable of anchoring to yeast membranes and/or walls in a stable manner by covalent or non-covalent interactions.
  3. 3. The yeast cell of claim 1, wherein the [ IFT ] is selected from the group consisting of c-myc-tag (SEQ ID NO:1)、 HA-tag (SEQ ID NO:2)、 Flag-tag (SEQ ID NO:3)、 His-tag (SEQ ID NO: 4)、 Strep-tag (SEQ ID NO:5)、 TC-tag (SEQ ID NO:6)、 S-tag (SEQ ID NO:7)、 AviTag (SEQ ID NO:8)、 Strep-tag2 (SEQ ID NO:9)。
  4. 4. The yeast cell of claim 1, further comprising one or more vectors encoding an Orthogonal Translation System (OTS) consisting of an aminoacyl-tRNA synthetase (aaRS) and a corresponding transfer RNA (tRNA) capable of inserting an unnatural amino acid at the TAG, TAA, TGA stop codon present in the expression vector encoding the fusion protein (I).
  5. 5. An expression vector encoding a fusion protein (I) as defined in claims 1-4.
  6. 6. The expression vector of claim 5, comprising a secretion signal located at a 5' position relative to fusion protein (I).
  7. 7. The expression vector of claims 5-6, further comprising an inducible promoter functionally linked to the fusion protein (I).
  8. 8. A cell population consisting of a plurality of yeast cells, wherein each cell is as defined in claims 1-4 and expresses on its surface a plurality of copies of a unique target macrocyclic peptide sequence, the amino acid sequence of which can vary within the cell population.
  9. 9. The cell population of claim 8, wherein the sequence diversity of the cysteine-enriched macrocyclic peptide of interest is at least 10 7 , preferably >10 8 , more preferably >10 9 .
  10. 10. A macrocyclic peptide library comprising a population of yeast cells according to claims 8 and 9, wherein different cells express different macrocyclic peptides of interest, preferably for each cell, wherein the macrocyclic peptides of interest are selected from ：(X) n C(X) m C(X) t 、(X) n C(X) m C(X) t C(X) y 、(X) n C(X) m C(X) t C(X) j C(X) y , wherein X, C, n, m, t, y, j are as defined in claim 1.
  11. 11. Use of a macrocyclic peptide library according to claim 10 in screening for biologically active macrocyclic peptide ligands.

Description

Macrocyclic peptide libraries displayed on the surface of yeast cells Technical Field The present invention relates to the generation of libraries of cysteine-enriched cyclic peptide sequences expressed on the surface of yeast cells. More specifically, the present invention provides a yeast cell-based macrocyclic peptide display system, a method of preparing the same, and a yeast cell population genetically modified to express a plurality of disulfide-tethered (disulfide-tethered) macrocyclic peptide ligands having different structures and amino acid sequences on the cell surface. Background Macrocyclic peptides are increasingly being demonstrated as a very valuable drug development molecular form (Ji, nielsen and Heinis 2024). Currently, there are about 80 peptide therapeutics on the global market, of which more than 40 are macrocyclic peptides, and the number of macrocyclic peptide drugs available per year is increasing year by year (Muttenthaler et al, 2021; fetse et al, 2023). Macrocyclic peptides have many advantageous properties that make them an extremely attractive model for the development of therapeutic drugs (Zhang and Chen 2022). They are capable of binding to macromolecular targets with high affinity and high selectivity. In addition, they often exhibit good proteolytic stability, in some cases even membrane permeability. Furthermore, they generally exhibit low inherent toxicity or antigenicity. In addition, macrocyclic peptides can be efficiently prepared by chemical synthesis and are easy to modify. In addition, the modular structure of the peptide can conveniently obtain hundreds of commercially available amino acid building units, and the process of developing various macrocyclic peptide variants with specific customization characteristics is simplified. All these properties make macrocyclic peptides advantageous, able to bridge the gap between small molecule drugs and large biological agents such as antibodies (Zhang and Chen 2022). Although many macrocyclic peptides still result from the exploration and development of naturally occurring peptides, recent technological advances and major breakthroughs in the field of molecular biology have paved the way for the development of "de novo" macrocyclic peptide ligands with specific desirable properties (Li, craven and Levine 2022). Indeed, the advent of powerful combinatorial techniques such as phage display (G.P. Smith and Petrenko 1997; deyle, kong and Heinis 2017) and mRNA display, greatly accelerated the generation of macrocyclic peptide ligands, enabling them to act against a variety of protein targets where no natural peptide ligand has yet been found. All these methods of directed evolution in vitro rely on the physical link between "phenotype" (expressed macrocyclic peptides) and "genotype" (coding DNA or RNA sequences). By applying such techniques, macrocyclic peptide ligands with the desired properties typically evolve following similar protocols involving the generation of large combinatorial libraries of random gene-encoded macrocyclic peptides, and multiple iterative selection, amplification and diversity cycles. In recent years, the development of innovative strategies for post-translational chemical and enzymatic modification has greatly expanded the number of macrocyclic peptide forms beyond classical disulfide tethered peptides that can be investigated by the combination techniques described above (Passioura et al, 2014; shalma et al, 2023; jaroszewicz et al, 2022; dotter et al, 2021). These advances, coupled with recent technological advances in screening programs and automation, the advent of new reagents and tools, and the maturation of new generation sequencing technologies, have made it possible to construct larger libraries and isolate macrocyclic peptide ligands with excellent binding properties that are capable of binding to increasingly challenging targets (t.p. Smith et al, 2023; stellwagen et al, 2019; sloth et al, 2023). Examples of macrocyclic peptides successfully found using in vitro display techniques include Merck & co. Oral PCSK9 inhibitor MK-0616 (Kingwell 2023) for the treatment of atherosclerotic cardiovascular diseases, and UCB recently obtained FDA approved drug zilucoplan (Mullard 2023) for the treatment of anti-acetylcholine receptor antibody positive adult systemic myasthenia gravis. Although these techniques have been demonstrated to be able to generate and screen extremely large libraries of sequence and structural diversity, enabling rapid identification of macrocyclic peptide ligands against virtually any target, they still rely on rather difficult to control procedures because it is difficult to monitor the performance of individual screening clones or populations during high throughput screening. Typically, the success of the screening activity will only be seen after a few weeks of work when isolated macrocyclic peptide molecules are identified. Furthermore, biophysical characterization of selec