WO2024133559A1 - Construction de vecteur pix modifiée - Google Patents

Construction de vecteur pix modifiée Download PDF

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Publication number
WO2024133559A1
WO2024133559A1 PCT/EP2023/087102 EP2023087102W WO2024133559A1 WO 2024133559 A1 WO2024133559 A1 WO 2024133559A1 EP 2023087102 W EP2023087102 W EP 2023087102W WO 2024133559 A1 WO2024133559 A1 WO 2024133559A1
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phage
pix
protein
coat protein
modified
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PCT/EP2023/087102
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English (en)
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Geir Åge LØSET
Gøril BERNTZEN
Benedikte Elisabeth HOBÆK
Nicolay Rustad NILSSEN
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Nextera As
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Definitions

  • the present invention relates to modified pIX filamentous phage coat proteins for phage display and phage particles displaying fusion proteins comprising such modified pIX proteins. More particularly, the present invention relates to vector constructs and nucleic acid molecules encoding said modified pIX phage coat proteins, preferably fused to proteins of interest, in order to display said proteins of interest on the surface of the phage as a pIX fusion protein.
  • phage display has become a powerful and efficient method for discovery and evolution of novel binding proteins.
  • the principle of combinatorial phage display technology is based on the genotype - phenotype linkage offered by the property that each virion will only display on its surface the very same proteins that are encoded by the genome encapsulated by its protein coat.
  • the phage particle itself is highly resistant to a variety of physiochemical conditions; hence phage display offers superior versatility in many selection regimes as compared to competing combinatorial technologies.
  • competing combinatorial technologies exist none show the high degree of versatility combined with the ease of use. Nonetheless, it is still challenging to screen for the desired phenotype following phage panning, and it is not given that the optimal binders are identified.
  • Phage display of heterologous polypeptides has been achieved using all five structural proteins of the filamentous phage coat, but pill- has gained the most widespread use.
  • pIX display has also been described and has been shown to have certain advantages over standard pill display in terms of efficacy in identifying desired target specific antibody candidates as well as yielding clones with superior biophysical properties (Hoydahl et al., 2016, Sci.Rep. 6, 39066).
  • the present inventors have now developed an improved pIX phage display system that utilizes an improved pIX expressing vector construct.
  • the improvement is in the form of providing a vector encoding a modified pIX phage coat protein in which rather than the full-length wild type pIX protein being provided, the vector encodes a modified pIX phage coat protein in which the methionine (M) residue at position 1 is replaced by an alternative amino acid residue.
  • M methionine
  • phage display in the form of improved antigen or target binding/reactivity or improved fusion protein display or functionality), or infectious phage particle production (infectious phage titre), or both, when compared to phage display using the wild-type pIX protein.
  • Such improvements are highly advantageous for phage display either in terms of the ability to successfully select and isolate binders at all, the quality and/or quantity of binders that can be selected, or in terms of the size of the phage display library that can be produced.
  • the most effective use of phage display as an engineering and discovery tool requires both the highest possible functional display in combination with the highest possible infective virion/phage particle production. This will ensure that the largest heterologous fusion protein library pool can be screened for the desired variant properties, thereby improving the prospects of identifying a protein with the desired properties.
  • the present invention manages to achieve this combination of properties.
  • phage display systems that enable high valency (HV) display, i.e. systems designed to maximise the number of pIX fusion proteins displayed on the surface of the phage when compared to the number of wild type (or non-fusion) pIX proteins displayed on the surface.
  • HV high valency
  • the present invention provides a vector construct comprising an open reading frame comprising a nucleic acid sequence encoding a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue.
  • the present invention provides a vector construct comprising a nucleic acid sequence encoding a pIX phage coat protein (a modified pIX phage coat protein) in which the methionine (M) residue at position 1 is replaced by an alternative amino acid residue.
  • the vectors of the invention are expression vectors or expression constructs, i.e. are generally comprised of nucleic acid sequences which enable the expression (protein synthesis) of desired encoded protein components in an appropriate host cell.
  • the vectors of the invention can be phage vectors or phagemid vectors (plasmids) the basic construction and components of which will be well known to a person skilled in the art and selected in order to achieve expression of phage proteins and packaging of phage particles in an appropriate host cell such that the heterologous or exogenous proteins (proteins of interest, POIs) fused to the modified pIX phage coat proteins of the invention are displayed on the surface of the phage particle.
  • phage vectors or phagemid vectors plasmids
  • phage particles are produced which contain desired POIs fused to the modified pIX phage coat protein of the invention and displayed on the surface of the phage particle, with the vector sequences or other nucleic acid sequences encoding the various phage components of the phage genome and also encoding the POI contained within the phage particle.
  • an appropriate host cell e.g. an appropriate prokaryotic host cell, such as an appropriate E. coli strain
  • the methionine (M) residue at position 1 of the pIX phage coat protein e.g. a wild-type or native pIX phage coat protein
  • the methionine (M) residue at position 1 of the pIX phage coat protein can be replaced or exchanged by any alternative amino acid residue, i.e. any amino acid residue that is not methionine (M).
  • the N-terminal methionine (M) residue of the pIX phage coat protein e.g. a wild-type or native pIX phage coat protein, can be replaced or exchanged by any alternative amino acid residue, i.e. any amino acid residue that is not methionine (M).
  • modified pIX phage coat proteins as used in the invention or as encoded by the constructs of the invention are referred to herein as modified pIX phage coat proteins or non-wild type pIX phage coat proteins of the invention.
  • the modified pIX phage coat proteins of the invention do not correspond to wild-type pIX phage coat proteins in terms of amino acid sequence.
  • the alternative amino acid residue which replaces the methionine (M) is selected from leucine (L), glycine (G), isoleucine (I), phenylalanine (F), tryptophan (W), tyrosine (Y), asparagine (N), glutamine (Q), glutamic acid (E), aspartic acid (D), proline (P), arginine (R), lysine (K), histidine (H), cysteine (C), serine (S), threonine (T), alanine (A), or valine (V).
  • the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T, or A.
  • the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, or H, or selected from L, G, I, C, S, T, or A.
  • the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, or H.
  • the alternative amino acid residue is selected from L, G, or I, more preferably is L or G, most preferably L.
  • any alternative amino acid residue can be used, the choice may also be guided by the desired outcome of the phage display process. For example, in many instances it will be desired to have as high phage production as possible combined with as high fusion protein functionality as possible. This would ensure the ability to cover the largest possible functional diversity in any fusion protein library, that should maximize the ability to retrieve and identify desired novel fusion proteins resulting from library selection. In this case, the results presented herein show that the exchange of the Methionine with Leucine (M1 L), Glycine (M1G) or Isoleucine (M 11) appears to translate into the optimal blend of these two disparate but connected features.
  • M1 L Methionine with Leucine
  • M1G Glycine
  • Isoleucine M 11
  • M1 E or M1 D represent appropriate alternative amino acids which give rise to very high target reactivity and that M1F, M1W, M1Y, M1N or M1Q would be other options.
  • M1 P, M1R, M 1 K or M 1 H would be further options.
  • the alternative amino acid residue is not R, K, D, S, A, V, T, C, H, P, E, Q, N, Y, W, F, I, G or L.
  • the alternative amino acid is not R. In some embodiments it is not K. In some embodiments it is not D. In some embodiments it is not S. In some embodiments it is not A. In some embodiments it is not V. In some embodiments it is not T. In some embodiments it is not C. In some embodiments it is not H. In some embodiments it is not P. In some embodiments it is not E. In some embodiments it is not Q. In some embodiments it is not N. In some embodiments it is not Y. In some embodiments it is not W. In some embodiments it is not F. In some embodiments it is not I. In some embodiments it is not G. In some embodiments it is not L.
  • position 2 of the modified pIX phage coat protein of the invention is S. In some embodiments, position 3 of the modified pIX phage coat protein is V. In some embodiments, position 4 of the modified pIX phage coat protein is L. In some embodiments, one or more, two or more, preferably all, of positions 2, 3, and 4 of the modified pIX phage coat protein is S, V and L, respectively. In some embodiments, position 2 of the modified pIX phage coat protein of the invention is not S. In some embodiments, position 3 of the modified pIX phage coat protein is not V. In some embodiments, position 4 of the modified pIX phage coat protein is not L.
  • one or more, two or more, preferably all, of positions 2, 3, and 4 of the modified pIX phage coat protein is not S, V and L, respectively.
  • position 16 is not C.
  • said modified positions are not M.
  • the modified pIX phage coat proteins of the invention have (or encode) an amino acid residue at position 1 of the pIX protein, but it is not methionine (M).
  • methionine (M) at position 1 of the pIX protein is replaced or exchanged for an alternative amino acid. It is not sufficient to merely delete or remove the methionine (M) at position 1.
  • modified pIX proteins or pIX fragments in which the methionine (M) at position 1 has been deleted or removed e.g.
  • pIX fragments containing or consisting of position 2 onwards e.g. position 2 to 32 for a full- length pIX protein, or pIX fragments in which the M at position 1 has been removed, are not encompassed by the invention.
  • vector constructs with a deleted methionine (M) at position 1 of the pIX protein do not show the same advantages and improved properties over wild-type pIX as the vector constructs of the invention.
  • open reading frame takes its standard art recognised meaning.
  • ORF open reading frame
  • ORF is used herein to refer to a span of a nucleic acid molecule, typically DNA, between a start and stop codon, or between a translation start and translation stop site.
  • Such ORFs typically encode a polypeptide, in this case a polypeptide which comprises a modified pIX phage coat protein of the invention as described herein.
  • Preferred ORFs encode a fusion protein of a POI and a modified pIX phage coat protein of the invention.
  • Appropriate start codons would be well known to a person skilled in the art.
  • a typical and exemplary start codon would be ATG encoding methionine.
  • the start codon would be positioned in the vector (or nucleic acid molecule) of the invention at an appropriate distance upstream of the sequence encoding the modified pIX phage coat protein in order for translation of the modified pIX phage coat protein to be initiated under appropriate conditions.
  • a POI is also encoded by the vector (or nucleic acid molecule) of the invention
  • the start codon is positioned at an appropriate distance upstream of, for example close to, or directly adjacent to, the sequence encoding the POI-modified pIX fusion in order for translation of the POI-modified pIX fusion protein to be initiated under appropriate conditions.
  • Appropriate stop codons would be well known to a person skilled in the art. Typical and exemplary stop codons would be TAA, TGA or TAG. One or more stop codons can be used.
  • vectors or nucleic acid molecules of the invention contain a single ORF.
  • pIX phage coat protein or “pIX protein” or “pIX phage protein” or “pIX coat protein”, etc., as used herein refers to a pIX protein originating from or derived from a filamentous phage, for example wild-type or native pIX filamentous phage coat protein sequences, or a pIX protein with a sequence which corresponds to the sequence of such a pIX protein.
  • Preferred filamentous phages from which the pIX protein is derived or the pIX protein corresponds to are M13, fd, or f1 phages.
  • any appropriate pIX protein can be used providing it has the ability to display a POI as a pIX fusion protein on the surface of a phage particle.
  • wild-type (or native) pIX proteins or wildtype like pIX proteins e.g. which comprise all the amino acids of wild-type (or native) pIX, but also incorporating one or more additional amino acids, e.g. in the form of conditional mutations, are used in some embodiments of the invention, e.g.
  • the pIX protein encoded by the vectors of the invention, or otherwise used in a fusion protein with a POI in order for the POI to be displayed on the phage surface corresponds to a pIX protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue.
  • M methionine
  • the modified pIX phage coat proteins encoded by the vectors of the invention comprise or consist of a full-length pIX phage coat protein providing that the methionine (M) residue at position 1 of the full-length pIX filamentous phage coat protein is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • Such full-length pIX phage coat proteins can typically have 32 amino acids.
  • the modified pIX phage coat proteins encoded by the vectors of the invention comprise or consist of the following amino acid sequence, which corresponds to the wild-type pIX protein from the VCSM13 helper phage (Genbank AY598820.1).
  • SEQ ID NO:1 providing that the methionine (M) residue at position 1 of the pIX filamentous phage coat protein (here SEQ ID NO:1) is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • nucleic acid sequence encoding this sequence for inclusion in the vectors of the invention is provided elsewhere herein as SEQ ID NO:2, again providing that the nucleic acid sequence encoding the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by a nucleic acid sequence encoding an alternative amino acid residue, e.g. as described elsewhere herein.
  • a yet further embodiment of the invention provides a vector construct of the invention, wherein the encoded modified pIX filamentous phage coat protein corresponds to the pIX coat protein from M13, fd or f1 phage, or a variant thereof, providing that the methionine (M) residue at position 1 is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • the encoded pIX protein comprises or consists of an amino acid sequence with a sequence identity of at least 60%, 65%, 70%, 75% or 80% to that of SEQ ID NO: 1 , such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, or 96 % identity, providing that the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue.
  • M methionine
  • position 1 of the pIX protein sequence should not be methionine (M).
  • Preferred alternative amino acid sequences for inclusion at position 1 are described elsewhere herein and can result in improvements in phage display, for example improved production of infectious phage particles (infectious phage titre) and/or improved functional protein display, e.g. improved POI/antibody/fusion protein display, e.g. improved antigen or target binding.
  • nucleic acid molecule encoding a variant of the modified pIX protein of the invention can for example comprise or consist of a nucleotide sequence with a sequence identity of at least 60%, 65%, 70%, 75% or 80% to that of SEQ ID NO: 2, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identity, providing that the methionine (M) residue encoded at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • M methionine
  • a yet further embodiment of the invention provides a vector construct of the invention, wherein the encoded modified pIX filamentous phage coat protein comprises SEQ ID NO:1 (MSVLVYSFASFVLGWCLRSGITYFTRLMETSS), or a sequence with at least 60%, 65%, 70%, 75% or 80% etc., identity to SEQ ID NO:1, providing that the methionine (M) residue at position 1 is replaced by an alternative amino acid residue, e.g. as described elsewhere herein. Exemplary and preferred % identity values are provided elsewhere herein.
  • modified pIX sequences e.g. variant modified pIX sequences, encoded by the vectors or nucleic acid molecules of the invention are sequences containing up to 12, e.g. up to 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 , altered amino acids in the pIX sequence, e.g. SEQ ID NO:1 , providing that the methionine (M) residue at position 1 of the pIX filamentous phage coat protein (e.g. SEQ ID NO:1) is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • M methionine
  • a further embodiment of the invention provides a vector construct of the invention, wherein the encoded modified pIX filamentous phage coat protein comprises SEQ ID NO:1 (MSVLVYSFASFVLGWCLRSGITYFTRLMETSS), or a sequence containing up to 12, e.g. up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 , altered amino acids in the pIX sequence, e.g. in SEQ ID NO:1, providing that the methionine (M) residue at position 1 is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • SEQ ID NO:1 MSVLVYSFASFVLGWCLRSGITYFTRLMETSS
  • modified pIX sequences or variants thereof should retain or have the functional ability to display a POI as a pIX fusion protein on the surface of a phage particle.
  • Functional C-terminal truncations or N-terminal fragments of SEQ ID NO:1 (or the variant sequences) or other pIX sequences could also be used providing that the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue, e.g. as described elsewhere herein, and that the ability to display a POI as a pIX fusion protein is retained.
  • fragments are not used.
  • full-length pIX proteins can be used, for example pIX proteins where all 32 amino acids (or variants thereof, e.g. as described elsewhere herein) are present, with the proviso that the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • M methionine
  • sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at the nucleotide level.
  • the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.
  • the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
  • Methods to determine the percentage identity of two amino acid sequences or of two nucleic acid sequences are well known and described in the art, and any of these may be used.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • Gapped BLAST may be utilised.
  • PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules.
  • sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
  • sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
  • the default settings with respect to e.g. “scoring matrix” and “gap penalty” may be used for alignment.
  • the BLASTN and PSI BLAST default settings may be advantageous. In calculating percent identity, only exact matches are counted.
  • the sequence encoding the modified pIX phage coat protein of the invention is linked, e.g. operably linked, to a sequence encoding a protein of interest (POI).
  • the vector construct, or the open reading frame (ORF) of the vector construct further comprises a sequence encoding a protein of interest (POI) linked, e.g. operably linked or fused, to the sequence encoding the modified pIX phage coat protein of the invention.
  • preferred vector constructs and nucleic acid molecules of the invention encode a POI- modified pIX fusion protein.
  • fusion protein is used herein to describe the functional joining of two or more protein components in the same polypeptide sequence or in the same open reading frame (ORF).
  • Such fusion proteins can also be described as genetic fusions as they are encoded by the same nucleic acid sequence (sometimes called a “fusion gene” or “fusion nucleotide sequence”).
  • fusion gene or “fusion nucleotide sequence”.
  • two (or more) protein components (or encoding nucleic acid sequences) can be directly adjacent to each other in such a fusion protein, equally the components can be joined by appropriate peptide spacers or linkers.
  • spacers or linkers can be important to allow each of the individual protein components to be expressed in a functional manner, e.g. allowing them to form the appropriate three-dimensional structure to perform or maintain their desired function.
  • a peptide spacer is generally included between the protein of interest (POI) and the modified pIX phage coat protein of the invention.
  • linkers or spacers need not be included, or may only be included in between some of the components.
  • the sequences encoding the POI can be fused to the sequences encoding the modified pIX phage coat protein of the invention with or without a spacer or linker sequence between the components. All these possibilities (i.e.
  • fusion proteins or encoding nucleic acids with or without spacer or linker sequences are still regarded as direct fusions or direct genetic fusions.
  • linker sequences may be included elsewhere in the vectors of the invention as appropriate, e.g. between other components of the vectors as discussed herein, for example between the VH and VL domains of an antibody POI or other POIs which involve or comprise two or more separate polypeptide components.
  • pIX fusion protein refers to a pIX protein (pIX phage coat protein, pIX filamentous phage coat protein), fused to an exogenous peptide/polypeptide, e.g. a protein of interest (POI).
  • modified pIX fusion protein refers to a modified pIX protein (modified pIX phage coat protein, modified pIX filamentous phage coat protein) of the invention fused to an exogenous peptide/polypeptide, e.g. a protein of interest (POI).
  • Preferred vectors of the invention thus comprise a sequence (a nucleic acid sequence) encoding a modified pIX phage coat protein of the invention fused (genetically fused) to a sequence encoding a POI (sometimes referred to herein as POI-modified pIX or POI-modified pIX fusion protein).
  • the POI and the modified pIX can be in any appropriate order or spacing in the vector providing that, once expressed and packaged into phage particles, a functional fusion protein between the POI and the modified pIX is formed wherein the modified pIX coat protein component of the fusion protein forms part of the phage coat and the POI is functionally expressed or displayed on the surface of the phage particle.
  • the POI part of the fusion protein is thus positioned in frame with the modified pIX coat protein part of the fusion protein.
  • This means that the POI and the pIX are expressed in the same polypeptide sequence (or as part of the same ORF), or, put another way, as a direct fusion.
  • the POI component of the fusion protein be positioned N-terminally (or at or near the N-terminus) of the modified pIX component of the fusion protein.
  • a yet further aspect of the invention provides a modified pIX filamentous phage coat protein of the invention.
  • such aspects of the invention provide a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • fusion proteins comprising said modified pIX phage coat proteins of the invention are provided, e.g. a fusion protein comprising a POI and a modified pIX phage coat protein of the invention.
  • such aspects of the invention provide a POI fused (preferably N-terminally) to a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • Nucleic acid molecules encoding such modified pIX filamentous phage coat proteins and fusion proteins are also provided.
  • nucleic acid sequences or nucleic acid molecules which can form part of the vectors of the invention or which comprise components of the vectors of the invention.
  • another aspect of the invention provides a nucleic acid molecule or nucleic acid sequence comprising an open reading frame comprising a nucleic acid sequence encoding a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue, e.g. as described elsewhere herein.
  • the present invention provides a nucleic acid molecule or nucleic acid sequence encoding the modified pIX phage coat protein of the invention or a fusion protein of the invention comprising a POI fused to a modified pIX phage coat protein of the invention.
  • one or more ribosome (ribosomal) binding sites are included in the vector constructs.
  • Such components can also be referred to as a translational initiation region (TIR).
  • the RBS sequence is located in the vector at an appropriate position for the RBS sequence to function.
  • the role of the RBS is to recruit a ribosome during the initiation of protein translation and thus is conveniently placed at an appropriate distance upstream from the start codon of the protein it is desired to translate, or upstream of the ORF for the protein it is desired to translate.
  • the RBS sequence is conveniently placed upstream of the sequence encoding the POI-modified pIX fusion protein.
  • a signal peptide is also part of the ORF, e.g.
  • the RBS sequence is conveniently also placed upstream of the sequence encoding the signal peptide (or upstream of the start codon of the ORF comprising the nucleic acid sequence encoding the signal peptide).
  • the RBS sequence is conveniently placed at an appropriate distance upstream of the sequence encoding the POI-modified pIX fusion protein.
  • the appropriate distance would be known or readily determined by a person skilled in the art depending on the RBS chosen. Exemplary distances might be seven or eight nucleotides from the ATG (or other) start codon, but this can vary.
  • the RBS/TIR sequence modulates the translation intensity (level of protein expression) of the sequences located downstream and different types of RBS can produce different levels of protein expression, for example weak or strong expression.
  • Weak or strong RBS/TIR sequences are well known in the art and can readily be selected by a skilled person depending on the level of protein expression desired.
  • a strong RBS facilitates or induces more translation (strong translation) as compared to a weak RBS.
  • Both weak and strong RBS sequences can be used in the vectors of the invention. In some embodiments, a weak RBS is used.
  • an RBS is included upstream (or 5’ or N-terminal to) to the start codon of the sequence encoding (or upstream, etc., of the start codon of the ORF comprising the nucleic acid sequence encoding) the POI- modified pIX fusion protein.
  • a preferred RBS for use in the present invention is a Shine Dalgarno (SD) sequence or a SD based sequence which can be included in the vector constructs. SD sequences are well known and described in the art and any of these may be used.
  • the core SD sequence is GAGG (SEQ ID NO:3) and other consensus sequences are GAGGAG (SEQ ID NO:4) or AGAGGAG (SEQ ID NO:5) or AGGAGAA (SEQ ID NO:6), for example comprising the sequence AGGAG (SEQ ID NO:7).
  • SD sequences comprising these core or consensus sequences can be used.
  • FIG. 3B An exemplary structure for a construct of the invention with a POI-modified pIX fusion protein is shown in Figure 3B.
  • the POI as shown in Figure 3B is an scFv antibody fragment, but this is just an example of a POI or library of POIs that could be used.
  • a sequence encoding a spacer or linker (typically a peptide spacer or linker) is included between the sequence encoding the POI and the sequence encoding the modified pIX phage coat protein.
  • sequences are typically synthetic or artificial (e.g. non-native or non-natural) linker or spacer sequences, e.g. sequences that do not encode functional proteins or protein domains.
  • Composite linker sequences can also be used.
  • linker or spacer sequences may include tag sequences such as c-Myc or a FLAG tag (e.g. DYKDDDDK; SEQ ID NO:8).
  • Full or full-length linker or spacer sequences are generally used, for example such sequences are generally not truncated sequences.
  • the inclusion of such a sequence can aid the folding of the connected proteins, in particular the N-terminal protein (here generally the POI), and thus the spacer or linker length can be adjusted as appropriate to enable the best or satisfactory functional folding of both components (i.e. the POI and the modified pIX).
  • Appropriate lengths could readily be determined by a person skilled in the art. However exemplary lengths would be between five and 15 amino acids (Weiss et al., 2000, Protein Sci. , 9:647-654), e.g. 6 to 10 amino acids.
  • a particular linker used in the present invention is AAAGSKDIR (SEQ ID NO:12).
  • a linker such as a GS linker, for example a linker with a certain number of GS repeats, e.g. G4S repeats, could be used.
  • such spacers or linkers form a distinct part of the vector or fusion protein than the modified pIX phage coat protein.
  • such spacers or linkers are not part of the modified pIX phage coat protein of the invention; the modified pIX phage coat protein of the invention is a distinct or separate component, e.g. there is a junction between the modified pIX phage coat protein and the upstream part of the vector.
  • any alternative amino acid residue used as a replacement at position 1 of the modified pIX phage coat protein of the invention is part of the pIX component (part) of the vector and is not part of the spacer or linker sequence (or any other part of the vector); the spacer or linker sequence (or the POI sequence) is a distinct or separate component.
  • Such distinct parts of the vector constructs are often separated by restriction enzyme sites or site-specific recombination sites.
  • a restriction enzyme site or site-specific recombination site is incorporated between the linker or spacer component (or other parts of the vector) and the modified pIX component.
  • preferred vectors of the invention can encode a protein of interest (POI) or targeting unit fused to the modified pIX phage coat protein.
  • POI protein of interest
  • Such embodiments allow the display of a POI, e.g. a targeting protein, on a modified pIX coat protein of the invention.
  • the use of the modified pIX phage coat proteins of the invention can result in improved display of the POIs.
  • the POI (and indeed any linker or spacer sequence placed between the POI and the modified pIX phage coat protein) is typically exogenous or heterologous.
  • an exogenous or heterologous protein what is meant is a protein or peptide not originally part of the relevant phage coat protein, e.g. the pIX protein, etc., which is fused (with or without any linker or spacer amino acids, which are also exogeneous or heterogeneous and thus not part or originally part of the relevant phage coat protein) to the modified pIX phage coat protein of the invention, e.g. fused to the N-terminal end of the modified pIX phage coat protein of the invention, e.g. fused to the N-terminal amino acid residue used as an alternative to the M residue at position 1 of the pIX phage coat protein.
  • Any protein of interest can be encoded in the vectors of the invention providing that it is suitable for display on a phage and in particular as a fusion with a pIX phage coat protein, for example a modified pIX phage coat protein of the invention.
  • POIs targeting molecules/targeting units or binding partners/binding proteins which can bind to other entities (targets/target entities, e.g. target proteins).
  • Some preferred examples of POIs would be antibodies or fragments thereof (e.g. Fab, scFv, nanobodies), MHC molecules (class I or class II), T cell receptors (TCRs), or non-lg derived binding proteins such as DARpins, Ankyrin family, fibronectin family, knottins, anticalins, etc., (Hosse et al., 2006, Protein Sci 15:14-27) and peptides.
  • nucleic acid molecules encoding these polypeptides can simply be positioned in the vectors such that a fusion protein with the modified pIX phage coat protein of the invention is produced.
  • the chosen type of POI is in the form of two or more polypeptide chains, e.g.
  • nucleic acid molecules encoding one of the polypeptides can be positioned in the vectors such that a fusion protein with the modified pIX phage coat protein of the invention is produced, and the other polypeptide chain(s) can be produced separately or independently.
  • the vectors of the present invention can be used for classical phage display in order to select binding partners (e.g. antibodies) for a particular target entity, e.g. target protein or target antigen.
  • a library of POIs can be expressed on phage particles as part of a fusion protein with the modified pIX phage coat protein of the invention and selected for binding to a target entity by standard and well-known techniques.
  • Another preferred component of the vector constructs of the invention is an appropriate promoter sequence in order to control the expression of the ORF comprising the modified pIX protein of the invention and fusion proteins containing said modified pIX protein.
  • Appropriate promoter sequences would be well known to a person skilled in the art and any of these could be used.
  • An exemplary promoter sequence might be a lac promoter which can for example be induced with IPTG.
  • Other promoters may include tac, arabB, or psp.
  • a signal sequence or signal peptide can be included in the ORF comprising the nucleic acid sequence encoding the modified pIX phage coat protein of the invention.
  • a signal sequence or signal peptide can be present or absent in the vectors or nucleic acid molecules of the invention. If present, then an appropriate location would readily be determined.
  • Such signal sequences are generally located upstream of (N-terminal of), but as part of the same ORF as, the POI-modified pIX fusion protein of the invention.
  • a signal sequence or signal peptide is not used or present.
  • Signal sequences or signal peptides can also sometimes be referred to as leader sequences or leader peptides.
  • the vectors e.g. the phage vectors or phagemid vectors (which can collectively be termed phage display vectors or constructs) may optionally additionally contain other appropriate components, for example origins of replication, inducible or non-inducible promoters/operators for initiating transcription, enhancers, termination sequences, antibiotic resistance genes and markers, sequences encoding chaperone proteins (e.g.
  • periplasmic chaperone proteins such as FkpA
  • signal sequences linkers, protease sites, general tags or reporter molecules, restriction enzyme or sitespecific recombination sites to enable cloning and other manipulations, e.g. for cloning appropriate POIs into the vectors of the present invention in an appropriate position to form a fusion protein with the modified pIX phage coat protein, primer binding sites to enable amplification of the constructs by e.g. PCR, or other desirable sequence elements, for example, DNA sequences to allow the discrimination between different libraries by e.g. PCR.
  • Appropriate sources and positioning of such additional components within the phage display constructs so that they perform their desired function would be well within the normal practice of a skilled person in the art.
  • nucleic acid molecules encoding the modified pIX phage coat proteins of the invention, or the fusion proteins of the invention which comprise said modified pIX phage coat proteins of the invention form yet further aspects.
  • the vectors of the invention are primarily used for phage display and can therefore be phagemid vectors or phage vectors.
  • the vector construct is a phagemid or a phage vector.
  • Phage display is a technique that is well known and described in the art.
  • G. P. Smith established a method to display polypeptides on the surface of filamentous phage, a virus that infects E. coli cells (Smith, G.P., 1985, Science 228, 1315-1317). Since then, so called phage display has evolved into a powerful technology for protein engineering and selection of peptides and proteins binding a specific target (Loset and Sandlie, 2012, Methods 58, 40-46).
  • the filamentous phage M13 is built from five different structural proteins.
  • Protein VIII (pVII I) is the major coat protein, and the particle is capped at one end by 5 copies of pill and pVI, and at the other end by 5 copies of pVII and pIX.
  • the particle infects F pilus+ E.coli by way of pill, and its ssDNA is injected into the bacterial cell.
  • phage DNA is replicated and transcribed, and new phage particles are assembled before nonlytic secretion into the growth medium.
  • phage display In phage display, a gene encoding a protein of interest (POI) is normally placed between a gene encoding a coat protein (often pill but here pIX) and its N-terminal signal sequence, to produce a POI-coat protein fusion, although in some embodiments of the present invention signal sequences are not present.
  • POI library or “library of phage particles” or similar refers to a collection of unique phages that differ in the amino acid sequence of the POI, and can be prepared by standard molecular cloning techniques. A library may well contain >1O 10 members, and can be used for selection of specific binders.
  • the present invention further provides phage or phage particles comprising the vectors or nucleic acid molecules of the invention and expressing a modified pIX filamentous phage coat protein or a modified pIX-fusion protein of the invention on the surface.
  • the phage particles may thus comprise a phage genome or phagemid, preferably a phagemid.
  • Such phage or phage particles can be any filamentous phage.
  • Preferred examples are Enterobacteria phage, for example M13, fd or f1 phages.
  • Another aspect of the present invention provides a library of phages/phage particles, e.g. filamentous phages, produced using and therefore comprising the vectors (or nucleic acid molecules) of the invention as described herein.
  • Said phages comprise fusion proteins of POIs with modified pIX phage coat proteins as described herein.
  • said filamentous phages display a POI or a library of POIs as fusions to the modified pIX phage coat protein of the invention.
  • each individual phage particle expresses/displays the same POI, but the presence of multiple particles expressing different POIs allows the display of multiple (or a library or a plurality of) different POIs.
  • a collection of diverse protein fusions e.g. diverse antibody fusions
  • a desired target in the form of a phage display library, e.g. an antibody phage display library.
  • a library is generally made up of a collection of either artificially or endogenously diversified proteins of interest, e.g. antibodies, fused to a phage capsid (here modified pIX), and these proteins of interest, e.g. antibodies, vary in their biophysical, biochemical and target binding properties.
  • Such libraries are then employed to identify those variants that harbour the property of interest through a cyclic process termed panning where each clone in the library competes with each other to enrich for the favourable variants.
  • a yet further aspect of the invention provides a library of phage particles, wherein the phage particles comprise the vectors (or nucleic acid molecules) of the invention as described herein, and wherein multiple different proteins of interest are expressed on the surface of the phage particles fused to a modified pIX phage coat protein of the invention.
  • POI-modified pIX fusion proteins of the invention can be encoded either in a complete phage genome by insertion of the sequences encoding the POI-modified pIX fusion protein into the phage genome (phage vector display), or on a phagemid (phagemid display).
  • a phagemid is a high copy number plasmid that can encode the POI-modified pIX fusion protein, and superinfection with a helper phage that provides the genetic material required for phage production, is required.
  • the coat protein that is utilized for POI display here the pIX phage coat protein
  • the helper phage encoded pIX protein e.g. a pIX protein that is not fused to a POI; a non-fused pIX protein
  • the phagemid encoded POI-pIX phage coat protein fusion here a POI-modified pIX fusion protein.
  • the new virions that are produced will then have a mixture of phagemid derived POI-modified pIX fusion proteins and helper phage derived pIX coat proteins (non-fused pIX coat proteins).
  • helper phage encoded pIX proteins/non-fused pIX proteins can be wild-type (or native), or wild-type like pIX proteins.
  • non-fused, e.g. wild-type or wild-type like, pIX phage proteins also generally need to be present, although in some embodiments of the present invention, no non-fused, e.g. no wild-type or wild-type like, form of the pIX coat protein is present.
  • the phage particles it is possible for the phage particles to be engineered to have one copy or to have multiple copies of the POI displayed on the modified pIX coat protein.
  • a phage genome system this can for example be achieved by modifying the phage genome to contain a sequence encoding (or an ORF comprising a sequence encoding) a POI-modified pIX fusion protein of the invention. If this is the only version/form of the pIX phage coat protein in the phage genome then multiple copies of the POI will be displayed on the modified pIX coat protein, and high valency (HV) display should be achieved as there will be no other form of the pIX phage coat protein to compete for surface display.
  • HV high valency
  • an alternative version/form of the pIX phage coat protein is provided in the system such that two versions of the pIX phage coat protein are present in the phage genome, e.g. by further modifying the phage genome to contain a sequence encoding (or an ORF comprising a sequence encoding) another pIX protein (non-fused pIX protein) as well as the modified-pIX phage coat protein of the invention, then the two forms of pIX will compete with each other for surface display and a mixture of POI-modified pIX fusion protein and non- fused pIX protein will be present on the surface, thereby achieving low valency (LV) display.
  • LV low valency
  • helper phage which is used and in preferred embodiments of the invention phage particles with multiple copies of the POI displayed on the modified pIX coat protein are used.
  • This can be achieved in any appropriate manner.
  • a modified type of helper phage for example a helper phage termed DeltaPhage, that allows high valency (HV) display on pIX.
  • HV high valency
  • modified helper phages contrast the use of normal helper phages such as M13K07, VCSM13, R408 or similar that only allows for low valency (LV) display.
  • the helper phage called DeltaPhage reported by Nilssen et al (Nilssen et al., 2012, Nucleic acids research, 40, e120; WO 2011/036555), has at least one (e.g. two) amber mutations inserted close to the pIX start codon, i.e. close to the codon encoding the methionine (M) residue at position 1 of the pIX phage coat protein, thereby conditionally inactivating (conditionally suppressing) the helper phage encoded pIX.
  • these amber mutations were placed between position 2 and position 3 of the pIX phage coat protein, i.e. between the residues S and V of the wild-type pIX phage coat protein.
  • other positions would be possible providing that said mutants would act to conditionally inactivate (conditionally suppress) the helper phage encoded pIX.
  • helper phage is then superinfected into a host cell (e.g. E. coli) transformed with a phagemid encoding a POI-pIX fusion, e.g. a POI-modified pIX fusion protein of the invention, then in a host cell which suppresses the amber mutation, such as an amber suppressor strain (e.g. a supE+ strain) of E coli, intermediate (low) valency display of the POI-pIX is seen, whereas in a host cell which does not suppress the amber mutation, such as an amber non-suppressor strain (e.g. a supE-/supE negative strain) of E.
  • a host cell e.g. E. coli
  • a host cell which suppresses the amber mutation such as an amber suppressor strain (e.g. a supE+ strain) of E coli
  • an amber non-suppressor strain e.g.
  • the vector construct is a phagemid vector which encodes a POI-modified pIX fusion protein of the invention and such a vector construct is used in combination with a helper phage which has a conditional mutation such that expression/production of the helper phage encoded pIX (non-fused pIX) phage protein can be controlled, which in turn can enable control of the number of POI-modified pIX fusion proteins on the surface of the phage.
  • the conditional mutation is not suppressed, e.g. when a non-suppressor strain of E coli, e.g. an amber non-suppressor strain, e.g.
  • helper phage encoded pIX should not be expressed/produced (will be suppressed) and only the POI-modified pIX should be expressed/produced resulting in only POI-modified pIX fusion proteins of the invention on the surface (high valency, HV, display).
  • conditional mutation is suppressed, e.g. when a suppressor strain of E coli, e.g. an amber suppressor strain, e.g.
  • helper phage encoded pIX will be produced/expressed resulting in a mixture of helper phage pIX (non-fused pIX) and POI-modified pIX fusion proteins on the surface (low valency, LV, display).
  • conditional mutations would be well known to a person skilled in the art and helper phage vectors can readily be designed, and appropriate host cells chosen, so that the expression of the helper phage encoded pIX is under control of the conditional mutation.
  • helper phage vectors can readily be designed, and appropriate host cells chosen, so that the expression of the helper phage encoded pIX is under control of the conditional mutation.
  • conditional mutations in the form of one or more suppressible stop codons e.g. amber mutations/amber stop codons, or other suppressible stop codons such as ochre or opal mutations/stop codons
  • suppressible stop codons e.g. amber mutations/amber stop codons, or other suppressible stop codons such as ochre or opal mutations/stop codons
  • HV high valency
  • a phagemid plus helper phage
  • any helper phage in which the pIX phage coat protein is lacking (e.g. has been deleted) or which does not produce a functional pIX phage coat protein (e.g. due to mutation or truncation) can be combined with a phagemid of the invention, i.e. a phagemid comprising a sequence encoding (or an ORF comprising a sequence encoding) a POI-modified pIX fusion protein of the invention, in order to achieve HV display.
  • high valency display and systems that allow high valency display
  • the modified-pIX vectors of the invention have been shown to be particularly effective and advantageous when combined with high valency display.
  • the modified-pIX vectors of the invention are compatible with low valency display, and systems that allow low valency display. Methods and systems to achieve low valency display would be well known to a person skilled in the art.
  • the modified-pIX vectors of the invention e.g. phagemid vectors, can be used with a helper phage such as DeltaPhage under suppressing conditions as discussed above.
  • the modified-pIX vectors of the invention can be used with more conventional helper phages encoding pIX (non-fused pIX) coat proteins, for example where the expression of the pIX coat protein is not subject to specific control or suppression, such as M13K07 or VCSM13, to achieve low valency display with a mixture of helper phage (non-fusion) pIX and POI-modified pIX fusion proteins on the surface.
  • low valency display and systems that allow low valency display, can be used with the modified-pIX vectors of the invention.
  • HV display has not traditionally been used to identify high affinity binders, as it is believed that the avidity effect through the display of multiple copies of the POI might compromise high affinity selection. Instead LV display is generally used to allow for high affinity binders to be identified.
  • modified pIX vectors of the present invention advantageously can be used in a HV display system to identify high affinity binders.
  • An HV display system has the further advantage of maximising the functional fraction of the phage particles, as more particles will have a POI-fusion protein meaning that more extensive functional diversity in the displayed POIs will be present, in turn meaning that finding a binder is more likely.
  • the ability of the HV display systems of the invention to combine an improved functional fraction with improved functional properties of the candidates displayed is highly advantageous, e.g. in terms of successfully identifying binders to a target of interest.
  • HV display refers to a phage display system which is designed to maximise the number of copies of a particular phage coat fusion protein, here the number of modified-pIX fusion proteins, displayed on the surface of the phage when compared to the number of wild-type (or non-fusion) pIX proteins displayed on the surface.
  • Such systems are thus designed such that all (theoretically all) of the copies of a particular phage coat protein, here the pIX phage coat protein, displayed on the surface of the phage should be POI fusion proteins, here POI- modified pIX fusion proteins.
  • the systems are designed such that 5 copies of the POI-modified pIX fusion protein can be displayed on each particle.
  • low valency (LV) display refers to a phage display system which is designed such that a mixture of coat protein fusion proteins, here modified- pIX fusion proteins, and wild-type (or non-fusion) pIX proteins are displayed on the surface of the phage.
  • Such systems are thus designed such that not all (theoretically not all), i.e. less than 5, e.g. 4, 3, 2, or 1, copies (or non-maximum copies or low copies, e.g.
  • POI fusion proteins here POI-modified pIX fusion proteins.
  • Such systems are typically set up to achieve an average of 1 , or less than 1, copy of a POI fusion protein per phage particle (although such systems can be set up to achieve a higher average number if desired).
  • systems set up to achieve an average of 1 , or less than 1 , copy of a POI fusion protein per phage particle many phage particles will not display a POI- modified pIX fusion protein at all.
  • the invention as described herein is designed for use in a prokaryotic system and not for example in a eukaryotic system.
  • appropriate host cells are prokaryotic cells and in particular bacterial cells.
  • Appropriate bacterial hosts for phage display which can be used to express the vectors and nucleic acid sequences of the invention and to package and produce phage particles would be well-known to a person skilled in the art and could be selected accordingly.
  • Preferred bacterial host cells are Gram negative bacteria such as strains of E. coli. Exemplary E. coli strains would include XL-1 blue, TG1, ER2738, AVBIOOFmkll’, MC1061, SS320,TGP10F’, and K91 K.
  • non-suppressor strains e.g. amber non-suppressor strains
  • are preferred examples of which are SS320, TOP10F’, AVBIOOFmkll’, MC1061 , and K91 K.
  • suppressor strains are used, e.g. XL-1 Blue, TG1 or ER2738.
  • phage often called bacteriophage
  • a filamentous bacteriophage, or filamentous phage is a phage with a single stranded DNA genome (ssDNA genome) which is packaged with phage coat proteins.
  • the secreted filamentous phage particle has phenotypically a filamentous structure. Filamentous bacteriophage or filamentous phage are preferred for use in the present invention.
  • phage or filamentous phage or filamentous bacteriophage as used herein encompasses both phage genome derived virions and phagemid-derived virions.
  • phagemid is a term of the art and refers to a type of cloning vector developed as a hybrid of the filamentous phage Ff and plasmids to produce a vector that can propagate as a plasmid, and also be packaged as single stranded DNA in viral particles.
  • a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques (e.g. transformation, electroporation).
  • infection of a bacterial host containing a phagemid with a 'helper' phage provides the necessary viral components to enable single stranded DNA replication and packaging of the phagemid DNA into phage particles.
  • helper phage is a term of the art and refers to a virus which helps a separate and unrelated defective virus, e.g. a phagemid, which in itself is not a phage genome or a functional virus, but merely a plasmid containing one or several elements derived from a phage genome (here at least a modified pIX protein of the invention), to reproduce by infecting the same host cell that is already occupied by the defective virus (e.g. phagemid) and providing the proteins which the defective virus (e.g. phagemid) is missing and needs to complete its life cycle and form virions, e.g. containing the phagemid.
  • a phagemid which in itself is not a phage genome or a functional virus, but merely a plasmid containing one or several elements derived from a phage genome (here at least a modified pIX protein of the invention)
  • helper phage for use in the present invention are described elsewhere herein and include M13K07 (Stratagene), Hyperphage (Progen Biotechnik GmbH), R408 (Agilent Technologies) and VCSM13 (Stratagene).
  • the helper phage may be a helper phage with a conditional (or suppressible) mutation as described herein e.g. the DeltaPhage helperphage as described herein and in the art, or Phaberge, or Ex-phage.
  • a phage display system comprising a vector (or nucleic acid molecule) of the invention.
  • Preferred phage display systems comprise a vector (or nucleic acid molecule) of the invention, e.g. a phagemid vector of the invention, and a helper phage, e.g. as described herein, e.g. a helper phage capable of expressing a pIX phage coat protein (e.g. a non-fused pIX phage coat protein).
  • Other preferred phage display systems of the invention comprise a vector (or nucleic acid molecule) of the invention, e.g.
  • a phagemid vector of the invention and a bacterial host cell, e.g. an E. coli host cell/strain.
  • a bacterial host cell e.g. an E. coli host cell/strain.
  • Appropriate host cells/strains are also described elsewhere herein and can be included as a component in all the phage display systems, kits, methods and uses described here.
  • Other preferred phage display systems comprise a vector (or nucleic acid molecule) of the invention, a helper phage, e.g. as described herein, e.g. a helper phage capable of expressing a pIX phage coat protein (e.g. a non-fused pIX phage coat protein) and a bacterial host cell, e.g. as described herein, e.g. an E. coli host cell/strain.
  • a helper phage e.g. as described herein, e.g. a help
  • a yet further embodiment of the invention provides a phage display system of the invention as described elsewhere herein, further comprising a helper phage and/or bacterial host cell strain, e.g. an E. coli host strain.
  • a helper phage and/or bacterial host cell strain e.g. an E. coli host strain.
  • the pIX phage coat protein encoded by the helper phage can complement or compete with the modified pIX phage coat protein encoded by the vector construct or nucleic acid molecule of the invention.
  • the pIX phage coat protein encoded by the helper phage can in effect provide additional copies of a pIX phage coat protein, e.g. additional copies of a non-fused pIX phage coat protein, e.g. additional copies of a functional pIX phage coat protein, which can be used to form the phage coat.
  • the pIX filamentous phage coat protein encoded by the helper phage is produced or expressed under the control of one or more conditional mutations, for example one or more suppressor mutations, e.g. as described elsewhere herein.
  • the suppressor mutation is a suppressible stop codon, preferably selected from the group consisting of amber, ochre and opal stop codons, more preferably an amber stop codon.
  • a preferred helper phage for use in such systems is DeltaPhage, details of which are described elsewhere herein and in the art.
  • Appropriate and preferred E coli host strains for use in such embodiments are suppressor strains, preferably an amber suppressor strain, more preferably XL-1 Blue, TG1 or ER2738.
  • the pIX phage coat protein encoded by the helper phage cannot complement or compete with the modified pIX phage coat protein encoded by the vector construct or nucleic acid molecule of the invention.
  • Such inability to complement or compete can result in any appropriate way. For example, such inability may arise because the pIX phage coat protein encoded by the helper phage is not functional, for example due to mutation or truncation, or because the pIX phage coat protein encoded by the helper phage is absent, for example due to deletion.
  • the pIX phage coat protein encoded by the helper phage is not produced or expressed, e.g. the pIX phage coat protein is produced or expressed under the control of one or more conditional mutations, for example one or more suppressor mutations, e.g. as described elsewhere herein, and the production or expression is suppressed.
  • the suppressor mutation is a suppressible stop codon, preferably selected from the group consisting of amber, ochre and opal stop codons, more preferably an amber stop codon, and the production or expression of the pIX phage coat protein is suppressed by using an appropriate bacterial host strain.
  • a preferred helper phage for use in such systems is DeltaPhage, details of which are described elsewhere herein.
  • Appropriate and preferred E coli host strains for use in such embodiments are nonsuppressor strains which do not allow production or expression of the pIX phage coat protein encoded by the helper phage, preferably an amber non-suppressor strain (or an ochre non-suppressor strain, or an opal non-suppressor strain), more preferably SS320 or TOP-1 OF’.
  • the vectors (or nucleic acid molecules) of the invention also find utility in phage display methods, i.e. can be used in phage display methods.
  • a yet further aspect of the invention provides a method for producing phage particles comprising the use of a vector construct or nucleic acid molecule of the invention or the use of a phage display system of the invention as described herein.
  • phage particles are typically produced by methods involving the steps of introducing the vector constructs of the invention, together with appropriate helper phages if necessary, into an appropriate bacterial host cell, examples of which are described elsewhere herein.
  • a yet further aspect of the invention thus provides a method of phage display comprising the steps of: a. providing a bacterial host cell/strain, e.g. an E coli host strain, comprising a vector construct of the invention wherein the open reading frame further comprises a sequence encoding a protein of interest fused to the sequence encoding the modified pIX phage coat protein of the invention, wherein expression of said vector construct results in production of a protein of interest-modified pIX fusion protein; b. providing a helper phage; and c. infecting said bacterial host cell/strain, e.g. said E coli host strain, with said helper phage under conditions such that said host strain produces phage particles that display said protein of interest-modified pIX fusion protein.
  • such methods can be used for high valency phage display.
  • said method is a method for high valency phage display, wherein the pIX filamentous phage coat protein encoded by the helper phage cannot complement the POI-modified pIX filamentous phage coat protein encoded by the vector construct of the invention.
  • the present invention provides a method for high valency phage display comprising the steps of: a) providing a non-suppressor bacterial host cell/strain, e.g. a non-suppressor E coli host strain, comprising a vector construct of the invention wherein the open reading frame further comprises a sequence encoding a protein of interest fused to the sequence encoding the modified pIX phage coat protein of the invention, wherein expression of said vector construct results in production of a protein of interest-modified pIX fusion protein; b) providing a helper phage, wherein expression of the pIX phage coat protein of the helper phage is under the control of one or more suppressor mutations; c) infecting said non-suppressor bacterial host cell/strain, e.g.
  • said E coli host strain with said helper phage under conditions such that the pIX phage coat protein encoded by the helper phage is not expressed or produced, such that said non-suppressor host cell/strain produces phage particles that display multiple copies of said protein of interest-modified pIX fusion protein.
  • such high valency display is preferred.
  • the methods of the invention can be used for low valency phage display.
  • said method is a method for low valency phage display, wherein the pIX filamentous phage coat protein encoded by the helper phage can complement the POI-modified pIX filamentous phage coat protein encoded by the vector construct of the invention.
  • the present invention provides a method for low valency phage display comprising the steps of: a) providing a suppressor bacterial host cell/strain, e.g. a suppressor E coli host strain, comprising a vector construct of the invention wherein the open reading frame further comprises a sequence encoding a protein of interest fused to the sequence encoding the modified pIX phage coat protein of the invention, wherein expression of said vector construct results in production of a protein of interest-modified pIX fusion protein; b) providing a helper phage, wherein expression of the pIX phage coat protein of the helper phage is under the control of one or more suppressor mutations; c) infecting said suppressor bacterial host cell/strain, e.g.
  • said E coli host strain with said helper phage under conditions such that the pIX phage coat protein encoded by the helper phage is expressed or produced, such that said suppressor host cell/strain produces phage particles that display single or low copies of said protein of interest-modified pIX fusion protein.
  • phage display in accordance with the invention and as described herein, e.g. high valency or low valency phage display, preferably a library of vector constructs of the invention are used which encode multiple proteins of interest. In other preferred embodiments said phage display methods are used for the selection of a protein which binds to a desired target molecule.
  • these proteins may be manufactured or produced, and if desired formulated with at least one pharmaceutically acceptable carrier or excipient.
  • Such manufactured molecules, or components, fragments, variants, or derivatives thereof, are also encompassed by the present invention.
  • these molecules may take the form of nucleic acids encoding said proteins, which nucleic acids may in turn be incorporated into an appropriate expression vector and/or be contained in a suitable host cell.
  • nucleic acid molecules encoding said proteins, or expression vectors containing said nucleic acid molecules form further aspects of the invention.
  • a yet further aspect of the invention provides a method of producing or manufacturing a protein (POI) comprising the steps of selecting the protein according to the methods of the invention as described herein, manufacturing or producing said protein, or a component, fragment, variant, or derivative thereof, and optionally formulating said manufactured protein with at least one pharmaceutically acceptable carrier or excipient.
  • said methods of the invention as described herein e.g. methods for selecting a protein, may further comprise the step of manufacturing or producing said protein, or a component, fragment, variant, or derivative thereof, and optionally formulating said manufactured or produced antibody with at least one pharmaceutically acceptable carrier or excipient.
  • Said variants or derivatives of protein may have at least 60, 70, 80, 90, 95 or 99% sequence identity to the original polypeptide from which they are derived.
  • kits comprising a vector (or nucleic acid molecule) of the invention or a kit comprising a phage display system of the invention as described above, for example comprising a phagemid of the invention and a helper phage, preferably a helper phage as described herein, e.g. a helper phage wherein the pIX phage coat protein is expressed under the control of a conditional (or suppressible) mutation as described herein, or a kit comprising a vector (or nucleic acid molecule) of the invention, e.g. a phagemid vector of the invention, and a bacterial host cell, e.g.
  • kits as described herein, e.g. a non-suppressor E. coli host strain.
  • the kit could also include the necessary instructions for use.
  • a kit comprising a phagemid of the invention, a helper phage, and a bacterial host cell as described herein is also provided.
  • Preferred vectors of the invention, helper phage and bacterial host cells for use in such kits are as described elsewhere herein.
  • Preferred vectors (or nucleic acid molecules) of the invention for inclusion in such kits could comprise a modified pIX phage coat protein vector of the invention as described herein, further comprising one or more cloning sites (e.g. a multiple cloning site) suitable for cloning in a POI which would then be fused to the modified pIX phage coat protein.
  • cloning sites e.g. a multiple cloning site
  • kits can thus comprise or consist of a collection of reagents for generating phage particles with a fusion protein of a POI to a modified pIX coat protein of the invention.
  • a kit could include one or more components selected from: other phagemids, helper phages, bacterial strains and instructions. Preferred options for such additional components are as described elsewhere herein.
  • a yet further aspect of the invention provides the use of a vector construct, a nucleic acid molecule, a phage display system or a kit of the invention to produce phage particles, or for use in phage display.
  • the present invention provides a method for producing phage particles (or a method of phage display), said method comprising the use of a vector construct, a nucleic acid molecule, a phage display system or a kit of the invention.
  • Such methods for producing phage particles typically involve the steps of introducing the vector constructs or nucleic acid molecules of the invention, together with appropriate helper phages if necessary, into an appropriate host cell, e.g. a bacterial host cell, examples of which are described elsewhere herein.
  • the phage particles of the invention as defined herein may also be used as molecular tools for in vitro applications and assays.
  • the particles may be used in any assay in which display of a POI on a pIX phage protein is desired.
  • phage particles of the invention also display a POI which can be a specific binding partner or targeting unit, e.g. an antibody etc., as described elsewhere herein, these can function as members of specific binding pairs or targeting reagents, and such phage particles can be used in any assay where the particular binding pair member or targeting unit is required.
  • a POI which can be a specific binding partner or targeting unit, e.g. an antibody etc., as described elsewhere herein, these can function as members of specific binding pairs or targeting reagents, and such phage particles can be used in any assay where the particular binding pair member or targeting unit is required.
  • yet further aspects of the invention provide a reagent that comprises phage particles of the invention as defined herein and the use of such phage particles as molecular tools, for example in in vitro assays.
  • the term "increase” or “improve” or “enhance” (or equivalent terms) as described herein includes any measurable increase or improvement when compared with an appropriate control.
  • Appropriate controls would readily be identified by a person skilled in the art and might include a level of a particular parameter as determined when a wild-type pIX phage coat protein is used in comparison to a modified pIX phage coat protein of the invention.
  • the increase, etc. will be significant, for example statistically significant, for example with a probability value of ⁇ 0.05, when compared to an appropriate control level or value. Methods of determining the statistical significance of differences are well known and documented in the art.
  • FIG. 1 Polyclonal phage ELISA and single-clone screening for OMV-reactivity.
  • A Normalized phage samples from RO and R3 outputs were analyzed for binding to OMV by ELISA. Phages displaying an irrelevant specificity (scFv anti-NIP) were included as control.
  • B Random single colonies after R3 were rescued to high valence (HV) display for all libraries. Samples were analyzed for OMV reactivity by ELISA and scored positive with a S/B (signal/background) ratio >3. The percentage of OMV positive clones within each library group is indicated. Supernatant from empty E. coli SS320 was included as a control.
  • FIG. 1 SDS-PAGE/Western blot analysis. Normalized amounts of phages (left) of the fully human antibody phagemid library (Hoydahl et al., 2016), and (right) a defined anti-phOx scFv phagemid control clone, were separated by 4-12% SDS PAGE followed by anti-pIX Western blot analysis probed with polyclonal rabbit anti-pIX serum. A M13K07 helper phage was included as a control (C). Both phagemid samples were packaged as either low valence (LV - rescued with M13K07) or high valence (HV - rescued with DeltaPhage) display. The pIXwt and scFv-pIX fusions are indicated.
  • FIG. 3 (A) Schematic illustration of the pV, pVII, pIX and pVIII encoding genomic region of M13 filamentous phage.
  • the pIX ORF has the start codon internal in the pVII ORF and is expressed as a complete protein without any post-translational processing.
  • FIG. 7 The Mix Anti-NIP scFv phage were separately rescued with DeltaPhage at LV (A) and HV (B) using in E. coli XL1-Blue and SS320, respectively, titrated and assessed for target binding to NIP-BSA in phage capture ELISA using serial dilution of each individual Mix variant. Thereafter, the variants were grouped according to their biochemical similarity and the averaged means ⁇ SD of the combined data of each individual binding curve within each group are shown as indicated. Notably, some of the samples produced very little phage and could only be tested at low titers.
  • FIG. 10 Low valent versus high valent display.
  • the M1L and wt anti-phOx and anti-NIP scFv phages were separately produced at LV and HV in E. coli XL1-blue or SS320, respectively. Phages were titrated and assessed for target binding to phOx- BSA (A) or NIP-BSA (B) in phage capture ELISA at serial dilutions. Performance of the LV and HV phages were compared with the standard low valence protocol using M13K07 rescue and E. coli XL1-blue (M1L_standard-LV and wt_standard-LV).
  • FIG. 11 Functional binding versus target concentration.
  • Anti-phOx and anti-NIP scFv displayed on pIXwt and M1L were produced at HV in E. coli SS320. Phages were titrated and assessed for target binding to reducing amount of phOx-BSA (A) or NIP- BSA (B) in phage capture ELISA using serial phage dilutions.
  • A phOx-BSA
  • B NIP- BSA
  • the NIP-specific scFv was prepared in E. coli SS320 (HV) using DeltaPhage helper phage and spiked into a target-irrelevant scFv at 1 :10 7 followed by 3 rounds of immobilized NIP-BSA panning. Forty randomly chosen single colonies were then packaged from each mock library before (RO) and after each round of selection (R1 - 3) and tested for target reactivity using an antigen-specific phage capture ELISA. Clones were regarded as positive if they exhibited at least 3-fold higher response than the background signal. The results are given as number of positive clones/total number of clones tested as indicated.
  • Figure 13 The fully human scFv antibody phage library displayed on pIXwt previously reported (Hoydahl et al., 2016) was reformatted to plX-M1 L and both libraries prepared at standard-LV and HV display from E. coli SS320 using the M13K07 and DeltaPhage helper phages, respectively. The apparent level of functionally folded scFv on the phages was then assessed by binding to the conformational specific superantigen protein L (pL) in serial dilutions of titrated phages using phage capture ELISA. A non-pL binding scFv control phage was included as control.
  • Figure 14 The fully human scFv antibody phage library displayed on pIXwt previously reported (Hoydahl et al., 2016) was reformatted to plX-M1 L and both libraries prepared at standard-LV and HV display from E. coli SS320 using the M13K07 and
  • the two indicated versions of the fully human scFv antibody phage library were used to individually select for pHLA-specific binders in three consecutive rounds (R1 - 3) of parallel panning using identical protocol towards two unrelated tumor associated antigen (TAA) specific pHLA targets.
  • TAA tumor associated antigen
  • R1 - 3 three consecutive rounds
  • RO unselected libraries
  • All samples were tested on both targets to serve as both specific screen and mutual negative controls on apparent specificity. The results for each sample are shown as the ratio in signal on the specific versus the unspecific target as an indirect measure of target specific enrichment.
  • An irrelevant scFv control phage was included as negative control (NC).
  • Random single clones from the R3 output selected towards pHLA TAA target 1 were expanded and phages produced using DeltaPhage rescue independent of the originating standard LV or HV form used in selection to maximize sensitivity in screening.
  • the phages were individually tested for binding to the matched (TAA target 1) and mis-matched (TAA target 2 and 3) pHLA targets in phage capture ELISA.
  • the clones are separated into their display capsid (A and B) and display version (standard- LV or HV) used in selection. The number of target specific clones in each version is indicated.
  • Example 1 Identification of a modified pIX version yielding improved antibody display
  • Single clones were packaged into 96-deep well plates using 400 pl culture medium for screening experiments or in 50 ml cultures for larger-scale expression. Brifely, clones were inoculated into YT-AG and cultured ON/37 °C. For 96-deep well expression 10 pl were transferred to new plates containing fresh medium and grown for 3 h/37 °C/600rpm before superinfection with 10 9 cfu DeltaPhage per well. For the larger-scale expression, cultures were reinoculated into fresh medium to ODeoonm of 0.05 and grown with rigorous shaking at 37 °C until ODeoonm reached 0.2 before superinfection with DeltaPhage or M13K07 at MO110.
  • ELISA plates were coated with serial dilutions of phOx-BSA or NIP-BSA in PBS starting at 5 pg/mL, incubated ON/4 °C and blocked with 4% skim milk powder in PBST for 1 h/RT. Phage were added at serial dilutions and incubated for 1 h/RT. Bound phage particles were detected with anti-M13-HRP (Amersham Biosciences, 1 :5,000). Phage samples and antibodies were diluted in PBST. Plates were developed with TMB solution and read at 450 nm using a microplate reader. Between each step, the plates were washed 3x with PBST.
  • Mouse anti-pl 11 (MoBiTec, 1 :5000) and anti-mouse IgG-HRP (1 :10.000) was used for pill detection.
  • pIX detection a polyclonal anti-pIX rabbit serum was generated by immunization using a peptide (N-CITYFTRLMETSS-C; SEQ ID NO:9) of the C-terminal pIX portion (AbMART).
  • the anti-pIX serum was used at 1 :2000 in combination with anti-rabbit IgG-HRP (1 :5000). Western blots were detected by reading chemiluminescent signals.
  • Phages displaying the NIP-specific scFv either on pIXwt or plX-M1 L were rescued with DeltaPhage from either of the two amber non-suppressor E. coli strains TOP10F’ or SS320, titrated and blended in a roughly 50/50 blend, followed by one round of selection on immobilized NIP-BSA. Briefly, ELISA wells (NUNC) were coated with 5 pg/mL phOx-BSA or NIP-BSA in PBS ON/4°C. Phages were incubated with antigen for 1.5 h at RT with agitation.
  • the scFv cassette was PCR amplified directly from the scFv-pIX library phages using 10 10 cfu AmpR as template, 0.25 pM each of forward 5' - ATTAAAGAGGAGAAATTAACCATGGCC-3' (SEQ ID NO: 10) and reverse 5' - TTTGGATCCAGCGGCCGC-3' (SEQ ID NO:11) biotinylated primers (Eurofins Genomics) containing Ncol and Notl RE-sites and 0.05 ll/rnl Phusion High- Fidelity DNA polymerase.
  • Amounts of 8.7x10 9 primary transformants were obtained which were scraped from the plate and rescued (see phage rescue and PEG/NaCI precipitation section).
  • the size of the library was limited to the size of the repertoire of the scFv-pIX library which was determined have a diversity of 3x10 8 . 2
  • Phagemid rescue was done by inoculating scraped material to ODeoo nm 0.05 in 2x YT supplemented with 30 p g/ml tetracycline, 100 p g/ml ampicillin and 0.1 M glucose (2x YT-TAG) and incubated with rigorous shaking at 37 °C until OD reached 0.1-0.2.
  • the cultures were superinfected with (DeltaPhage for plX_M1 L Library, R1 , R2 and R3 or M13K07 for R3 only) at MOI20 and incubated with gentle shaking at 37 °C for 60 min, followed by rigorous shaking for 30 min, before centrifugation and medium replacement to 2x YT supplemented with 100 p g/ml ampicillin and 50 p g/ml kanamycin (2x YT-AK) and incubation further 7 h at 30 °C. Phage particles were purified and concentrated by 2x PEG/ NaCI precipitation and resuspended in PBS and cfu was determined by spot titration. 1
  • ELISA plates were coated with 5 pg/mL protein L (pL) in PBS and incubated ON/4 °C and blocked with 2% skim milk powder in PBST for 1 h/RT. Phage samples were added in serial dilutions and incubated 2h/RT. Bound phage particles were detected with an anti-M13 antibody (generated by immunization of chickens with M13 bacteriophages by Norwegian antibodies) conjugated to HRP. Phage samples and antibodies were diluted in PBST. Plates were developed with TMB solution and read at 450 nm using a microplate reader. Between each step, the plates were washed 3x with PBST.
  • Antigen concentration was decreased 10-fold in each round, and washing stringency increased from 8xPBST + 2xPBS in R1 to 13xPBST + 2xPBS in R2 and 18xPBST + 2xPBS in R3.
  • the phage samples were heat challenged for 15 min at 65°C, before used in panning. Tubes were briefly vortexed between each wash. 4% (w/v) non-fat skim milk powder (PBSM) or 2% (w/v) bovine serum albumin (essentially fatty acid free) was used as blocking reagents in alternating selection rounds. Elution was performed by 10 min incubation with 0.5 ml 0.5% trypsin followed by E. coli infection using half of the eluate. Infected colonies were scraped and rescued (see phage rescue and PEG/NaCI precipitation section). A small sample of the infected culture was removed for determination of output.
  • PBSM non-fat skim milk powder
  • bovine serum albumin essentially fatty acid free
  • Single clones were packaged into 96-deep well plates as described. 4 Briefly, clones were inoculated into YT-AG and cultured ON/37 °C/600 rpm. 10 pl were transferred to new plates containing fresh medium and grown for 3 h before superinfection with 10 9 cfu DeltaPhage per well. Plates were incubated at 37 °C/30 min with gentle agitation, and further 30 min with vigorous shaking before cells were pelleted and resuspended in 50 l 2x YT-AK and phage were packaged ON/30 °C. 100 pL cleared supernatants were used for screening in ELISA.
  • ELISA plates were coated with 5 pg/mL NeutrAvidin in PBS and incubated ON/4 °C and blocked with 5% skim milk powder in PBST for 1 h/RT. Biotinylated pH LA variants were captured for 1 h/RT, followed by addition of phage. Bound phage particles were detected with anti-M13-HRP (Amersham Biosciences, 1:5,000). pHLA, phage samples and antibodies were all diluted in PBST. Plates were developed with TMB solution and read at 450 nm using a microplate reader. Between each step, the plates were washed 3x with PBST.
  • HV pIX display both outperformed LV and HV pill display in successful antibody identification ( Figure 1 C), as well as consistently identified the strongest target binders 1 .
  • the combined results of these analyses of the anti-phOx scFv clearly single out the M 1 L variant as the most favorable variant that both preserves an overall good virion production both at LV and HV, as well as exhibiting a clear improvement in target reactivity that must be assigned to a pronounced reduction in scFv devoid pIX protein component.
  • the results also strongly suggest that it is the native /V-terminal pIX Methionine that allows for this pIX off-product to be produced, and that the effect at large can be abolished by changing this particular residue.
  • Antibody phage display has a primary objective to be used in antibody engineering and discovery where collection of diverse antibody fusions with differing properties are displayed and selected on target in the form of an antibody library 15 .
  • These antibodies will inevitably have varying intrinsic efficiency in display and the human anti-phOx scFv represents a rather well-behaving unit in this context due as it is derived from multiple rounds of optimization using phage display engineering 16 .
  • the unmodified pIX and M1V variants were consistently in the least reactive bin.
  • the M1 L and M1 I variants were also consistently in the bin with the highest target reactivity across both LV and HV form.
  • effective use of phage display as an engineering and discovery tool requires both the highest possible functional display in combination with the highest possible infective virion production. This will ensure that the largest heterologous fusion protein library pool can be screened for the desired variant properties.
  • the M1 L variant consistently gave strongest target reactivity with both scFvs and both in LV and HV display. The difference was again greatest with the HV version of the anti-NIP scFv. Moreover, the M13K07-rescued phages were clearly subordinate in target reactivity for both scFvs and compared with both the LV and HV versions. Interestingly, the differences between the unmodified pIX and M1 L also appeared equalized when using M13K07 pointing to the use of DeltaPhage as a key helper phage reagent to disclose the underlying disparate profiles.
  • Example 2 Improved antibody discovery through use of M1 L modified pIX display
  • Example 1 we identified that the native /V-terminal Methionine of the pIX capsid in filamentous phage display could be beneficially exchanged with alternative amino acids, and in particular Leucine, to obtain improved heterologous fusion protein display.
  • alternative amino acids and in particular Leucine
  • one of the primary applications of phage display is as a combinatorial engineering and discovery tool 15 ’ 18 .
  • phage display used for antibody discovery a library is made up by a collection of either artificially or endogenously diversified antibodies fused to a phage capsid, and these antibodies vary in their biophysical, biochemical and target binding properties.
  • the two scFv variants were prepared in the HV version by DeltaPhage rescue from either the amber non-suppressor E. coli strains SS320 or TOP10F’.
  • the phages were mixed at a tentatively 1 :1 blend and panned against immobilized target (NIP-BSA) followed by sequencing of random single clones before and after panning to disclose if one version was preferentially enriched at the expense of the alternative clone.
  • This library has an estimated diversity of about 3x 10 8 unique antibody clones and hence serves as a suitable source for discovery of potentially new antibody specificities for further drug development. Care was taken to preserve the original antibody repertoire and based on transformation frequency sequence analysis the new library was estimated to have about the same diversity as prior to reformatting.
  • M1E or M1 D may represent a choice which gives very high target reactivity (M1 F, M1W or M1Y, or M1N or M1Q would be other options; M1P, M1R, M1 K or M1H would be further options).
  • M1C or M1S or M1T or M1A may represent appropriate choices in this scenario.

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Abstract

La présente invention concerne une construction de vecteur comprenant un cadre de lecture ouvert comprenant une séquence d'acide nucléique codant pour une protéine de revêtement de phages filamenteux pIX modifiée dans laquelle le résidu de méthionine (M) à la position 1 de la protéine de revêtement de phages filamenteux pIX est remplacé par un résidu d'acide aminé alternatif. L'invention concerne également des particules de phages ou un système d'affichage de phages comprenant lesdites constructions de vecteur, ainsi que des procédés d'affichage de phages et des kits.
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